AU2005203018A1 - Exonuclease-mediated nucleic acid reassembly in directed evolution - Google Patents

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Exonuclease-mediated nucleic acid reassembly in directed evolution The following statement is a full description of this invention, including the best method of performing it known to us:

EXONUCLEASE-MEDIATED

SNUCLEIC ACID REASSEMBLY IN DIRECTED EVOLUTION FIELD OF THE INVENTION 00 This invention relates to the field of protein engineering. More specifically, this relates to a directed evolution method for preparing a polynucleotides C 10 encoding polypeptide, which method comprises the step of generating site-directed mutagenesis optionally in combination with the step ofpolynucleotide C'l chimerization, the step of selecting for potentially desirable progeny molecules, including by a process termed end-selection (which may then be screened further), and the step of screening the polynucleotides for the production of polypeptide(s) having a useful property.

In a particular aspect, the present invention is relevant to enzymes, particularly to thermostable enzymes, and to their generation by directed evolution.

More particularly, the present invention relates to thermostable enzymes which are stable at high temperature and which have improved activity at lower temperatures.

BACKGROUND

Harvesting the full potential of nature's diversity can include both the step of discovery and the step of optimizing what is discovered. For example, the step of discovery allows one to mine biological molecules that have industrial utility.

However, for certain industrial needs, it is advantageous to further modify these enzymes experimentally to achieve properties beyond what natural evolution has provided and is likely to provide in the near future.

The process, termed directed evolution, of experimentally modifying a biological molecule towards a desirable property, can be achieved by mutagenizing one or more parental molecular templates and idendifying any desirable molecules among the progeny molecules. However, currently available technologies used in directed evolution have several shortfalls. Among these shortfalls are: 1) Site-directed mutagenesis technologies, such as sloppy or low-fidelity PCR, are ineffective for systematically achieving at each position (site) along a polypeptide sequence the full (saturated) range of possible 0_ mutations all possible amino acid substitutions).

Cr 2) There is no relatively easy systematic means for rapidly analyzing the large amount of information that can be contained in a molecular sequence and in the potentially colossal number or progeny molecules that could be N 10 conceivably obtained by the directed evolution of one or more molecular templates.

3) There is no relatively easy systematic means for providing comprehensive empirical information relating structure to function for molecular positions.

4) There is no easy systematic means for incorporating internal controls in certain mutagenesis chimerization) procedures.

There is no easy systematic means to select for specific progeny molecules, such as full-length chimeras, from among smaller partial sequences.

Molecular mutagenesis occurs in nature and has resulted in the generation of a wealth of biological compounds that have shown utility in certain industrial applications. However, evolution in nature often selects for molecular properties that are discordant with many unmet industrial needs. Additionally, it is often the case that when an industrially useful mutations would otherwise be favored at the molecular level, natural evolution often overrides the positive selection of such mutations when there is a concurrent detriment to an organism as a whole (such as when a favorable mutation is accompanied by a detrimental mutation). Additionally still, natural evolution is slow, and places high emphasis on fidelity in replication.

Finally, natural evolution prefers a path paved mainly by beneficial mutations while tending to avoid a plurality of successive negative mutations, even though such O negative mutations may prove beneficial when combined, or may lead through a circuitous route to final state that is beneficial.

Directed evolution, on the other hand, can be performed much more rapidly and aimed directly at evolving a molecular property that is industrially desirable 00 where nature does not provide one.

SAn exceedingly large number of possibilities exist for purposeful and random tcombinations of amino acids within a protein to produce useful hybrid proteins and their corresponding biological molecules encoding for these hybrid proteins, i.e., DNA, RNA. Accordingly, there is a need to produce and screen a wide variety of such hybrid proteins for a desirable utility, particularly widely varying random proteins.

The complexity of an active sequence of a biological macromolecule polynucleotides, polypeptides, and molecules that are comprised of both polynucleotide and polypeptide sequences) has been called its information content which has been defined as the resistance of the active protein to amino acid sequence variation (calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function).

Proteins that are more sensitive to random mutagenesis have a high information content.

Molecular biology developments, such as molecular libraries, have allowed the identification of quite a large number of variable bases, and even provide ways to select functional sequences from random libraries. In such libraries, most residues can be varied (although typically not all at the same time) depending on compensating changes in the context. Thus, while a 100 amino acid protein can contain only 2,000 different mutations. 20100 sequence combinations are possible.

Information density is the IC per unit length of a sequence. Active sites of enzymes tend to have a high information density. By contrast, flexible linkers of information in enzymes have a low information density.

Current methods in widespread use for creating alternative proteins in a library format are error-prone polymerase chain reactions and cassette O mutagenesis, in which the specific region to be optimized is replaced with a Ssynthetically mutagenized oligonucleotide. In both cases, a substantial number of Smutant sites are generated around certain sites in the original sequence.

c Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. In a mixture of fragments of unknown sequence, error-prone PCR can be used to mutagenize the mixture. The published error-prone PCR protocols suffer from a low processivity of the polymerase. Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene. This inability limits the practical application of error-prone PCR. Some computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the large-scale block changes that are required for continued and dramatic sequence evolution. Further, the published error-prone PCR protocols do not allow for amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application. In addition, repeated cycles of error-prone PCR can lead to an accumulation of neutral mutations with undesired results, such as affecting a protein's immunogenicity but not its binding affinity.

In oligonucleotide-directed mutagenesis, a short sequence is replaced with a synthetically mutagenized oligonucleotide. This approach does not generate combinations of distant mutations and is thus not combinatorial. The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization. Mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round followed by grouping them into families, arbitrarily choosing a single family, and reducing it to a consensus motif. Such motif is resynthesized and C N reinserted into a single gene followed by additional selection. This step process Sconstitutes a statistical bottleneck, is labor intensive, and is not practical for many rounds of mutagenesis.

00 Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful O for single cycles of sequence fine tuning, but rapidly become too limiting when O they are applied for multiple cycles.

Another limitation of error-prone PCR is that the rate of down-mutations grows with the information content of the sequence. As the information content, library size, and mutagenesis rate increase, the balance of down-mutations to upmutations will statistically prevent the selection of further improvements (statistical ceiling).

In cassette mutagenesis, a sequence block of a single template is typically replaced by a (partially) randomized sequence. Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences library size). This eliminates other sequence families which are not currently best, but which may have greater long term potential.

Also, mutagenesis with synthetic oligonucleotides requires sequencing of individual clones after each selection round. Thus, such an approach is tedious and impractical for many rounds of mutagenesis.

Thus, error-prone PCR and cassette mutagenesis are best suited, and have been widely used, for fine-tuning areas of comparatively low information content.

One apparent exception is the selection of an RNA ligase ribozyme from a random library using many rounds of amplification by error-prone PCR and selection.

In nature, the evolution of most organisms occurs by natural selection and sexual reproduction. Sexual reproduction ensures mixing and combining of the genes in the offspring of the selected individuals. During meiosis, homologous chromosomes from the parents line up with one another and cross-over part way along their length, thus randomly swapping genetic material. Such swapping or shuffling of the DNA allows organisms to evolve more rapidly.

0_ In recombination, because the inserted sequences were of proven utility in a c homologous environment, the inserted sequences are likely to still have substantial information content once they are inserted into the new sequence.

S 10 The term Applied Molecular Evolution means the application of an evolutionary design algorithm to a specific, useful goal. While many different library formats for AME have been reported for polynucleotides, peptides and proteins (phage, lacI and polysomes), none of these formats have provided for recombination by random cross-overs to deliberately create a combinatorial library.

Theoretically there are 2,000 different single mutants of a 100 amino acid protein. However, a protein of 100 amino acids has 20100 possible sequence combinations, a number which is too large to exhaustively explore by conventional methods. It would be advantageous to develop a system which would allow generation and screening of all of these possible combination mutations.

Some workers in the art have utilized an in vivo site specific recombination system to generate hybrids of combine light chain antibody genes with heavy chain antibody genes for expression in a phage system. However, their system relies on specific sites of recombination and is limited accordingly. Simultaneous mutagenesis of antibody CDR regions in single chain antibodies (scFv) by overlapping extension and PCR have been reported.

Others have described a method for generating a large population of multiple hybrids using random in vivo recombination. This method requires the recombination of two different libraries of plasmids, each library having a different selectable marker. The method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).

In vivo recombination between two homologous, but truncated, insect-toxin genes on a plasmid has been reported as a method of producing a hybrid gene. The 00 in vivo recombination of substantially mismatched DNA sequences in a host cell o having defective mismatch repair enzymes, resulting in hybrid molecule formation has been reported.

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SUMMARY OF THE INVENTION SThis invention relates generally to the field of nucleic acid engineering and correspondingly encoded recombinant protein engineering. More particularly, the invention relates to the directed evolution of nucleic acids and screening of clones containing the evolved nucleic acids for resultant activity(ies) of interest, such c nucleic acid activity(ies) &/or specified protein, particularly enzyme, activity(ies) of C interest.

C 10 This invention relates generally to a method of: 1) preparing a progeny generation molecule (including a molecule that is comprised of a polynucleotide sequence, a molecules that is comprised of a polypeptide sequence, and a molecules that is comprised in part of a polynucleotide sequence and in part of a polypeptide sequence), that is mutagenized to achieve at least one point mutation, addition, deletion, &/or chimerization, from one or more ancestral or parental generation template(s); 2) screening the progeny generation molecule preferably using a high throughput method for at least one property of interest (such as an improvement in an enzyme activity or an increase in stability or a novel chemotherapeutic effect); 3) optionally obtaining &/or cataloguing structural &/or and functional information regarding the parental &/or progeny generation molecules; and 4) optionally repeating any of steps 1) to 3).

In a preferred embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, comprising: subjecting a starting or parental polynucleotide set to an in vitro exonucleasemediated reassembly process so as to produce a progeny polynucleotide set; whereby the exonuclease-mediated reassembly process is exemplified, in a nonlimiting fashion, by subjection to a 3' exonuclease treatment, such as treatment with exonuclease III, which acts on 3' underhangs and blunt ends, to liberate 3'-terminal but not 5'-terminal nucleotides from a starting double stranded polynucleotide, leaving a remaining strand that is partially or completely free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner; whereby the exonuclease-mediated reassembly process is further exemplified, in a nonlimiting fashion, by subjection to a 5' exonuclease treatment, such as treatment c. with red alpha gene product, that acts on 5' underhangs to liberate nucleotides from a starting double stranded polynucleotide, leaving a remaining strand that is partially or completely free of its original partner so that, if 00 desired, the remaining strand may be used to achieve hybridization to another 0 partner, 0 whereby the exonuclease-mediated reassembly process is further exemplified, in a non-limiting fashion, by subjection to an exonuclease treatment, such as treatment with Mung Bean Nuclease or treatment with S 1 Nuclease or treatment with E.coli DNA Polymerase, that acts on overhanging ends, including on unhybridized ends, to liberate terminal nucleotides from an unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but hybridized end to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated end; and whereby the exonuclease-mediated reassembly process is also exemplified by a dual treatment, that can be performed, for example, non-simultaneously, with both an exonuclease that liberates terminal nucleotides from underhanging ends or blunt ends as well as an exonuclease that liberates terminal nucleotides from overhanging ends such as unhybridized ends.

In a preferred aspect of this embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, wherein the step of(a) subjecting a starting or parental polynucleotide set to an in vitro exonuclease-mediated reassembly process so as to produce a progeny polynucleotide set; is comprised of: subjecting a starting or parental polynucleotide set to a 3' exonuclease treatment that acts on 3' underhangs and blunt ends, to liberate 3'-terminal but not terminal nucleotides; whereby said 3' exonuclease is exemplified, in a non-limiting fashion, by treatment with an exonuclease, such as exonuclease III, to liberate 3'-terminal but not terminal nucleotides from a starting double stranded polynucleotide, leaving a Sremaining strand that is partially or completely free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to _another partner.

00 In another preferred aspect of this embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, wherein the step of subjecting a starting or parental polynucleotide set to an in vitro exonuclease-mediated reassembly process so as to produce a progeny S 10 polynucleotide set; is comprised of: subjecting a starting or parental polynucleotide set to a 5' exonuclease treatment that acts on 5' underhangs to liberate 5'-terminal nucleotides; whereby said 5' exonuclease is exemplified, in a non-limiting fashion, by treatment with an exonuclease, such as red alpha gene product, to liberate nucleotides from a starting double stranded polynucleotide, leaving a remaining strand that is partially or completely free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner.

In yet another preferred aspect of this embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, wherein the step of subjecting a starting or parental polynucleotide set to an in vitro exonuclease-mediated reassembly process so as to produce a progeny polynucleotide set; is comprised of: subjecting a starting or parental polynucleotide set to an exonuclease treatment that liberates terminal nucleotides from nucleic acid overhangs; whereby said treatment is exemplified, in a non-limiting fashion, by subjection to an exonuclease treatment, such as treatment with Mung Bean Nuclease or treatment with S Nuclease or treatment with E.coli DNA Polymerase, that acts on overhanging ends, including on unhybridized ends, to liberate nucleotides from an unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but 0 hybridized end to facilitate polymerase-based extension and/or ligase- C mediated ligation of the treated end.

In yet another preferred aspect of this embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, wherein the 00 step of subjecting a starting or parental polynucleotide set to an in vitro 0 exonuclease-mediated reassembly process so as to produce a progeny c) Spolynucleotide set; is comprised of: t subjecting a starting or parental polynucleotide set to a 3' exonuclease treatment that acts on 3' underhangs and blunt ends, to liberate 3'-terminal but not terminal nucleotides; and (ii) subjecting a starting or parental polynucleotide set to an exonuclease treatment that liberates terminal nucleotides from nucleic acid overhangs; whereby the exonuclease-mediated reassembly process is comprised of a dual treatment, that can be performed, for example, non-simultaneously, with both an exonuclease that liberates terminal nucleotides from underhangs or blunt ends as well as an exonuclease that liberates terminal nucleotides from overhangs such as unhybridized ends.

In yet another preferred aspect of this embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide, wherein the step of subjecting a starting or parental polynucleotide set to an in vitro exonuclease-mediated reassembly process so as to produce a progeny polynucleotide set; is comprised of: subjecting a starting or parental polynucleotide set to a 5' exonuclease treatment that acts on 5' underhangs to liberate 5'-terminal nucleotides; and (ii) subjecting a starting or parental polynucleotide set to an exonuclease treatment that liberates terminal nucleotides from nucleic acid overhangs; whereby the exonuclease-mediated reassembly process is comprised of a dual treatment, that can be performed, for example, non-simultaneously, with both an exonuclease that liberates terminal nucleotides from underhangs or blunt ends as well as an exonuclease that liberates terminal nucleotides from overhangs such as unhybridized ends.

In another preferred embodiment, this invention provides a method for producing a mutagenized progeny polynucleotide having at least one OC desirable property comprised of the step of: c subjecting a starting or parental polynucleotide set to an in vitro exonucleasemediated reassembly process so as to produce a progeny polynucleotide set; and N 10 subjecting the progeny polynucleotide set to an end selection-based screening and enrichment process, so as to select for a desirable subset of the progeny polynucleotide set; whereby the above steps can be performed iteratively and in any order and in combination; whereby the end selection-based process creates ligation-compatible ends; whereby the creation of ligation-compatible ends is optionally used to facilitate one or more intermolecular ligations, that are preferably directional ligations, within members of the progeny polynucleotide set so as to achieve assembly &/or reassembly mutagenesis; whereby the creation of ligation-compatible ends serves to facilitate ligation of the progeny polynucleotide set into an expression vector system and expression cloning; whereby the expression cloning of the progeny polynucleotide set serves to generate a polypeptide set; whereby the generated polypeptide set can be subjected to an expression screening process; and whereby expression screening of the progeny polypeptide set provides a means to identify a desirable species, e.g. a mutant polypeptide or alternatively a polypeptide fragment, that has a desirable property, such as a specific enzymatic activity.

0 In another preferred embodiment, this invention provides a method for CN generating a mutagenized progeny polynucleotide from a collection of Sprogenitor polynucleotides, comprising: a) annealing a poly-binding nucleic acid strand to two mono-binding nucleic acid strands to generate an annealed heteromeric complex of nucleic acid strands; oO wherein the poly-binding nucleic acid strand and the two mono-binding nucleic acid strands are each derived from a different molecular species in said collection of progenitor polynucleotides; wherein the said collection of progenitor polynucleotides is preferably comprised of nonidentical though possibly related progenitor polynucleotides, as exemplified by a collection of genes encoding dehalogenases; and wherein the poly-binding nucleic acid strand to two mono-binding nucleic acid strands each have at least a 7 nucleotide-long sequence of identity to the progenitor polynucleotides from which it is derived; and b) subjecting the unhybridized single-stranded ends of the annealed mono-binding nucleic acid strands in the heteromeric complex to an exonuclease treatment that degrades said unhybridized ends; whereby the annealment of working poly-binding and mono-binding strands derived from nonidentical polynucleotides thus allows one to generate a chimerization of said nonidentical polynucleotides; whereby, in a library of said annealed complexes of nucleic acid strands, many component strands have unhybridizable ends that are suboptimal or not serviceable for priming polymerase-based extension; and whereby the exonuclease treatment removes such unhybridizable ends to convert the annealed complexes of nucleic acid strands into better primers for polymerase-based extension.

In a preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, further comprising the step of: c) subjecting the annealed heteromeric complex to polymerase-based extension.

SIn another preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, further comprising the step of: d) subjecting the annealed nucleic acid strands to a ligase treatment; whereby subjection to ligase treatment is exemplified by subjection to T4 DNA 00 Ligase treatment to achieve intermolecular ligation between the two annealed Smono-binding strands, which thus become covalently linked forming a Schimerized strand.

In yet another preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, further comprising the step of: e) separating the poly-binding nucleic acid strand from the ligated mono-binding nucleic acid strands; whereby the separation of a poly-binding nucleic acid strand from ligated monobinding nucleic acid strands to which it is annealed can be achieved, for example, by either denaturation or by exposure to an enzymatic activity that selectively acts on the poly-binding nucleic acid strands.

In yet another preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, further comprising the step of: f) generating a nucleic acid strand that is complementary to the ligated monobinding nucleic acid strand; whereby the resultant product is comprised of a double stranded mutagenized progeny polynucleotide.

In yet another preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, further wherein the mutagenized progeny polynucleotide is a gene C or gene pathway.

In yet another preferred aspect of this embodiment, this invention provides a method for generating a mutagenized progeny polynucleotide from a collection of OO progenitor polynucleotides, further comprising: expressing the generated O mutagenized progeny polypeptide in a suitable host; whereby said expression leads O to the generation of a product of the polypeptide that can be detected by expression t screening.

O In a preferred embodiment, there is generated from a parent polynucleotide template) in what is termed "codon site-saturation mutagenesis" a progeny generation of polynucleotides, each having at least one set of up to three contiguous point mutations different bases comprising a new codon), such that every codon (or every family of degenerate codons encoding the same amino acid) is represented at each codon position. Corresponding to and encoded by this progeny generation of polynucleotides, there is also generated a set of progeny polypeptides, each having at least one single amino acid point mutation. In a preferred aspect, there is generated in what is termed "amino acid site-saturation mutagenesis" one such mutant polypeptide for each of the 19 naturally encoded polypeptide-forming alpha-amino acid substitutions at each and every amino acid position along the polypeptide. This yields for each and every amino acid position along the parental polypeptide a total of 20 distinct progeny polypeptides including the original amino acid, or potentially more than 21 distinct progeny polypeptides if additional amino acids are used either instead of or in addition to the 20 naturally encoded amino acids Thus, in another aspect, this approach is also serviceable for generating mutants containing in addition to &/or in combination with the 20 naturally encoded polypeptide-forming alpha-amino acids other rare &/or not naturallyencoded amino acids and amino acid derivatives. In yet another aspect, this approach is also serviceable for generating mutants by the use of- in addition to 004671139 S &/or in combination with natural or unaltered codon recognition systems of suitable hosts altered, mutagenized, &/or designer codon recognition systems (such as in a host cell with one or more altered tRNA molecules).

SIn yet another aspect, this invention relates to recombination and more specifically to a method for preparing polynucleotides encoding a polypeptide by a method of in vivo re- 0O assortment of polynucleotide sequences containing regions of partial homology, assembling the polynucleotides to form at least one polynucleotide and screening the polynucleotides for the production ofpolypeptide(s) having a useful property.

OIn yet another preferred embodiment, this invention is serviceable for analyzing and to cataloguing with respect to any molecular property an enzymatic activity) or combination of properties allowed by current technology the effects of any mutational change achieved (including particularly saturation mutagenesis). Thus, a comprehensive method is provided for determining the effect of changing each amino acid in a parental polypeptide into each of at least 19 possible substitutions. This allows each amino acid in a parental polypeptide to be characterized and catalogued according to its spectrum of potential effects on a measurable property of the polypeptide.

In another aspect, the method of the present invention utilizes the natural property of cells to recombine molecules and/or to mediate reductive processes that reduce the complexity of sequences and extent of repeated or consecutive sequences possessing regions of homology.

The present invention also provides a method for generating hybrid polynucleotides encoding biologically active hybrid polypeptides with enhanced activities. There has been provided, in accordance with one aspect of the invention, a method for introducing polynucleotides into a suitable host cell and growing the host cell under conditions that produce a hybrid polynucleotide.

O In another aspect of the invention, the invention provides a method for c screening for biologically active hybrid polypeptides encoded by hybrid Spolynucleotides. The present method allows for the identification of biologically active hybrid polypeptides with enhanced biological activities.

00 Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, 0that the detailed description and the specific examples, while indicating preferred tt embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

In a specific embodiment, this invention provides method for producing and isolating a library of progeny polunucleotides having at least one desirable property comprised of the steps of: subjecting a starting or parental polynucleotide set to a mutagenesis process so as to produce a progeny polynucleotide set; and subjecting the progeny polynucleotide set to an end selection-based screening and enrichment process, so as to select for a desirable subset of the progeny polynucleotide set; whereby the above steps can be performed iteratively and in any order and in combination, whereby the end selection-based process creates ligation-compatible ends, whereby the creation of ligation-compatible ends is optionally used to facilitate one or more intermolecular ligations, that are preferably directional ligations, within members of the progeny polynucleotide set so as to achieve assembly &/or reassembly mutagenesis, whereby the creation of ligation-compatible ends serves to facilitate ligation of the progeny polynucleotide set into an expression vector system and expression cloning, V whereby the end selection-based screening and enrichment process allows one to produce a library of progeny polynucleotides generated by a mutagenesis process, include non-stochastic polynucleotide sitesaturation mutagenesis (Gene Site Saturation Mutagenesis T M and non-stochastic polynucleotide reassembly (GeneReassemblyM), 00 whereby the expression cloning of the progeny polynucleotide set serves to generate a full-length polypeptide set, whereby the generated polypeptide set can be subjected to an expression screening process, and whereby expression screening of the progeny polypeptide set provides a means to identify a desirable species, e.g. a mutant polypeptide or alternatively a polypeptide fragment, that has a desirable property, such as a specific enzymatic activity.

In another specific embodiment, this invention provides a method for producing and isolating a polypeptide having at least one desirable property comprised of the steps of: subjecting a starting or parental polynucleotide set to a mutagenesis process so as to produce a progeny polynucleotide set; and subjecting the progeny polynucleotide set to an end selection-based screening and enrichment process, so as to select for a desirable subset of the progeny polynucleotide set; whereby the above steps can be performed iteratively and in any order and in combination, whereby the end selection-based process creates ligation-compatible ends, whereby the creation of ligation-compatible ends is optionally used to facilitate one or more intermolecular ligations, that are preferably directional ligations, within members of the progeny polynucleotide set so as to achieve assembly &/or reassembly mutagenesis, 0 whereby the end selection-based screening and enrichment process allows c one to produce a library of progeny polynucleotides generated by a Smutagenesis process, include non-stochastic polynucleotide sitesaturation mutagenesis (Gene Site Saturation Mutagenesis

T

and non-stochastic polynucleotide reassembly (GeneReassembly), 00 whereby the expression cloning of the progeny polynucleotide set serves to generate a full-length polypeptide set, c Swhereby the creation of ligation-compatible ends serves to facilitate ligation to of the progeny polynucleotide set into an expression vector system and expression cloning, whereby the generated polypeptide set can be subjected to an expression screening process, and whereby expression screening of the progeny polypeptide set provides a means to identify a desirable species, e.g. a mutant polypeptide or alternatively a polypeptide fragment, that has a desirable property, such as a specific enzymatic activity.

In a specific aspect of this embodiment, this invention provides the immediately preceding methods, wherein the mutagenesis process of step is comprised of a process, termed saturation mutagenesis, for generating, from a codon-containing parental polypeptide template, a progeny polypeptide set in which a full range of single amino acid substitutions is represented at each amino acid position, comprising the steps of: subjecting a working codon-containing template polynucleotide to polymerase-based amplification using a degenerate oligonucleotide for each codon to mutagenized, where each of said degenerate oligonucleotides is comprised of a first homologous sequence and a degenerate triplet sequence, so as to generate a set of progeny polynucleotides; wherein said degenerate triplet sequence is selected from the group consisting of i) N,N,N; ii) N,N,G/T; iii) N,N,G/C; iv) N,N,C/G/T; v) N,N,A/G/T; vi) N,N,A/C/T; vii) N,N,A/C/G; and viii) any degenerate codon that encodes all 20 amino acids; and 004671139 subjecting said set of progeny polynucleotides to recombinant expression such 0 Sthat polypeptides encoded by the progeny polynucleotides are produced; whereby the above steps can be performed iteratively and in any order and in combination, and 00 5 whereby, said method provides a means for generating all 20 amino acid changes Sat each amino acid site along a parental polypeptide template, because the degeneracy of Sthe triplet sequence includes codons for all 20 amino acids.

O In a specific aspect of this embodiment, this invention further provides the immediately preceding methods, wherein the mutagenesis process of step is comprised of a process, [0 termed synthetic ligation gene reassembly or simply synthetic ligation gene reassembly.

In another aspect, the present invention provides a method for producing a set of mutagenized progeny polynucleotides comprising: subjecting a set of partially double-stranded polynucleotides to in vitro exonuclease treatment, wherein the exonuclease is a 5' exonuclease or a 3' exonuclease, and subjecting the set of polynucleotides to non-stochastic extension and/or ligation, thereby producing a set of mutagenized progeny polynucleotides.

In another aspect, the present invention provides a method for producing a set of mutagenized progeny polynucleotides comprising: subjecting a set of partially double-stranded polynucleotides to in vitro exonuclease treatment, wherein the partially double-stranded polynucleotide comprises at least one poly-binding nucleic acid, and subjecting the set of polynucleotides to non-stochastic extension and/or ligation, thereby producing a set of mutagenized progeny polynucleotides.

004671139 ^t In a further aspect, the present invention provides a method for producing a plurality of mutagenized polynucleotides having at least one desirable property, comprising: subjecting a plurality of partially double-stranded polynucleotides to an exonuclease-mediated recombination process, thereby producing a plurality of mutagenized progeny polynucleotides, and 00 0(b) subjecting the mutagenized progeny polynucleotides to an end selection and 0screening process, so as to select one or more of the plurality of mutagenized progeny t) polynucleotides having at least one desirable property.

In yet another aspect, the present invention provides a method for generating a mutagenized progeny polynucleotide from a collection of progenitor polynucleotides, comprising: annealing a first single-stranded polynucleotide to at least two other singlestranded polynucleotides to generate a heteromeric complex of at least three polynucleotides, wherein the first polynucleotide and the two other polynucleotides are non-identical, but related and derived from the collection of progenitor polynucleotides, and wherein the first polynucleotide is partially hybridized to the two other polynucleotides, subjecting the single-stranded ends of the heteromeric complex to an exonuclease treatment that degrades the single-stranded ends of the heteromeric complex, and subjecting the heteromeric complex to non-stochastic polymerase-based extension and/or ligation, thereby generating a mutagenized progeny polynucleotide from the collection of progenitor polynucleotides.

In a further aspect, the present invention provides a method for producing in vitro a plurality of polynucleotides having at least one desirable property, said method comprising: subjecting a plurality of progenitor polynucleotides to an exonuclease-mediated recombination process so as to produce a plurality of progeny polynucleotides; and 004671139 subjecting the progeny polynucleotides to an end selection-based screening and 0 Senrichment process, so as to select one or more of the progeny polynucleotides having at least one desirable property.

SIn another aspect, the present invention provides a method of producing a plurality of mutant polypeptides having at least one desirable property said method comprising: 00 subjecting a plurality of progenitor polynucleotides to an exonuclease-mediated 0 recombination process so as to produce a plurality of progeny polynucleotides; introducing the progeny polynucleotides into a host cell so as to cause expression of a plurality of mutant polypeptides having an end selection marker; and subjecting the mutant polypeptides to an end selection-based screening so as to select one or more having at least one desirable property.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

As used herein, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS j Figure 1 shows the activity of the enzyme exonuclease HI. This is an exemplary enzyme that can be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks. The asterisk indicates that the enzyme o acts from the 3' direction towards the 5' direction of the polynucleotide substrate.

SFigure 2 shows an exemplary application of the enzyme exonuclease III in Sexonuclease-mediated polynucleotide reassembly. Shown also is the combined use of in vivo "repair" by transforming a suitable host Escherichia, Pseudomonas, Steptomyces, or Bacillus) and utilizing the host's repair mechanism to provide further diversity by generating a library of cloned mutagenized progeny nucleic acids and preferably polypeptides expressed by such nucleic acids) that can be analyzed by expression screening.

Figure 3 shows the generation of a poly-binding nucleic acid strand. In this case, the generated strand is of the same length as the parental template, but is not methylated.

Dpn I treatment can thus be used to select for the generated strand. Although not shown, the template as well as the generated product can be part of a vector (linear or circular).

Figure 4 shows the use of exonuclease treatment as a means to liberate 3' and terminal nucleotides from the unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but hybridized end to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated end.

Shown also is the combined use of in vivo "repair" by transforming a suitable host Escherichia, Pseudomonas, Steptomyces, or Bacillus) and utilizing the host's repair mechanism to provide further diversity by generating a library of cloned mutagenized progeny nucleic acids and preferably polypeptides expressed by such nucleic acids) that can be analyzed by expression screening.

0 Figure 5 shows the use of exonuclease-mediated nucleic acid reassembly in an CI example in which one methylated poly-binding nucleic acid strand is annealed to Sseveral unmethylated mono-binding nucleic acid strands. The annealed nucleic acid strands form heteromeric nucleic acid complexes and are subjected to exonuclease treatment as a means to liberate 3' and 5'-terminal nucleotides from the oO unhybridized single-stranded ends of a plurality of annealed nucleic acid strands in Sthe heteromeric nucleic acid complexes, leaving shortened but hybridized ends to 0 facilitate polymerase-based extension and/or ligase-mediated ligation of the treated Sends. Treatment with DpnI is then serviceable for selecting against the generated 0 10 annealed mono-binding nucleic acid strands that are unmethylated and chimeric in nature.

Figure 6 shows the use of exonuclease-mediated nucleic acid reassembly in an example in which a plurality of methylated poly-binding nucleic acid strand are annealed to several unmethylated mono-binding nucleic acid strands. The annealed nucleic acid strands form heteromeric nucleic acid complexes that are subjected to exonuclease treatment as a means to liberate 3' and 5'-terminal nucleotides from the unhybridized single-stranded ends of a plurality of annealed nucleic acid strands in the heteromeric nucleic acid complexes, leaving shortened but hybridized ends to facilitate polymerase-based extension and/or ligase-mediated ligation of the treated ends. Treatment with DpnI is then serviceable for selecting against the generated annealed mono-binding nucleic acid strands that are unmethylated and chimeric in nature.

DEFINITIONS OF TERMS SIn order to facilitate understanding of the examples provided herein, certain frequently occurring methods and/or terms will be described.

oO The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, an array of spatially localized compounds a VLSIPS Speptide array, polynucleotide array, and/or combinatorial small molecule array), n biological macromolecule, a bacteriophage peptide display library, a bacteriophage 0 10 antibody scFv) display library, a polysome peptide display library, or an extract made form biological materials such as bacteria, plants, fungi, or animal (particular mammalian) cells or tissues. Agents are evaluated for potential activity as antineoplastics, anti-inflammatories or apoptosis modulators by inclusion in screening assays described hereinbelow. Agents are evaluated for potential activity as specific protein interaction inhibitors an agent which selectively inhibits a binding interaction between two predetermined polypeptides but which doe snot substantially interfere with cell viability) by inclusion in screening assays described hereinbelow.

An "ambiguous base requirement" in a restriction site refers to a nucleotide base requirement that is not specified to the fullest extent, i.e. that is not a specific base (such as, in a non-limiting exemplification, a specific base selected from A, C, G, and but rather may be any one of at least two or more bases.

Commonly accepted abbreviations that are used in the art as well as herein to represent ambiguity in bases include the following: R G or A; Y C or T; M A or C; K G or T; S G or C; W Aor T; H Aor C or T; B G or T or C; V G or C or A; D G or A or T; N A or C or G or T.

The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH 2 and a carboxyl group (-COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds. The "twenty naturally encoded polypeptide-forming alpha-amino acids" are Sunderstood in the art and refer to: alanine (ala or arginine (arg or asparagine C' (asn or aspartic acid (asp or cysteine (cys or gluatamic acid (glu or E), Z glutamine (gin or glycine (gly or histidine (his or isoleucine (ile or I), leucine (leu or lysine (lys or methionine (met or phenylalanine (phe or proline (pro or serine (ser or threonine (thr or tryptophan (trp or W), 00 tyrosine (tyr or and valine (val or V).

O The term "amplification" means that the number of copies of a polynucleotide is increased.

0 The term "antibody", as used herein, refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')z, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, Harlow and Lane, supra), and are described further, as follows.

An Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

An (Fab') 2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent Sreduction. A (Fab') 2 fragment is a dimer of two Fab' fragments, held Stogether by two disulfide bonds.

S(4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

00 M An single chain antibody is a genetically engineered single N, chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible

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polypeptide linker.

A molecule that has a "chimeric property" is a molecule that is: 1) in part homologous and in part heterologous to a first reference molecule; while 2) at the same time being in part homologous and in part heterologous to a second reference molecule; without 3) precluding the possibility of being at the same time in part homologous and in part heterologous to still one or more additional reference molecules. In a non-limiting embodiment, a chimeric molecule may be prepared by assemblying a reassortment of partial molecular sequences. In a non-limiting aspect, a chimeric polynucleotide molecule may be prepared by synthesizing the chimeric polynucleotide using plurality of molecular templates, such that the resultant chimeric polynucleotide has properties of a plurality of templates.

The term "cognate" as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example, but not limitation, in the human genome the human CD4 gene is the cognate gene to the mouse 3d4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T cell activation through MHC class II-restricted antigen recognition.

A "comparison window," as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may O be compared to a reference sequence of at least 20 contiguous nucleotides and C wherein the portion of the polynucleotide sequence in the comparison window may Scomprise additions or deletions gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a 00 comparison window may be conducted by the local homology algorithm of Smith S(Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol, O 1981; Smith and Waterman, JMol Biol, 1981; Smith et al, JMolEvol, 1981), by tt the homology alignment algorithm ofNeedleman (Needleman and Wuncsch, O 10 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection, and the best alignment resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

As used herein, the term "complementarity-determining region" and "CDR" refer to the art-recognized term as exemplified by the Kabat and Chothia CDR definitions also generally known as supervariable regions or hypervariable loops (Chothia and Lesk, 1987; Clothia et al, 1989; Kabat et al, 1987; and Tramontano et al, 1990). Variable region domains typically comprise the aminoterminal approximately 105-115 amino acids of a naturally-occurring immunoglobulin chain amino acids 1-110), although variable domains somewhat shorter or longer are also suitable for forming single-chain antibodies.

"Conservative amino acid substitutions" refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and Stryptophan; a group of amino acids having basic side chains is lysine, arginine, and N. histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are :valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

00 oO The term "corresponds to" is used herein to mean that a polynucleotide 3 sequence is homologous is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference "TATAC" and is complementary to a reference sequence "GTATA." The term "degrading effective" amount refers to the amount of enzyme which is required to process at least 50% of the substrate, as compared to substrate not contacted with the enzyme. Preferably, at least 80% of the substrate is degraded.

As used herein, the term "defined sequence framework" refers to a set of defined sequences that are selected on a non-random basis, generally on the basis of experimental data or structural data; for example, a defined sequence framework may comprise a set of amino acid sequences that are predicted to form a B-sheet structure or may comprise a leucine zipper heptad repeat motif, a zinc-finger domain, among other variations. A "defined sequence kernal" is a set of sequences which encompass a limited scope of variability. Whereas a completely random sequence of the 20 conventional amino acids can be any of (20)10 sequences, and a pseudorandom 10-mer sequence of the 20 conventional amino acids can be any of (20)10 sequences but will exhibit a bias for certain residues at certain positions and/or overall, a defined sequence kernal is a subset of sequences if each residue position was allowed to be any of the allowable 20 conventional amino acids (and/or allowable unconventional amino/imino acids). A defined sequence kernal generally comprises variant and invariant residue positions and/or comprises C variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), and the like, either segmentally or over the entire length of the individual selected library member sequence. Defined sequence kernels can refer to either amino acid sequences or polynucleotide sequences. Of oO illustration and not limitation, the sequences (NNK)io and (NNM)o, wherein N Srepresents A, T, G, or C; K represents G or T; and M represents A or C, are defined sequence kernels.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 ug ofplasmid or DNA fragment is used with about 2 units of enzyme in about 20 tLI of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to jig of DNA are digested with 20 to 250 units of enzyme in a larger volume.

Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a gel to isolate the desired fragment.

"Directional ligation" refers to a ligation in which a 5' end and a 3' end of a polynuclotide are different enough to specify a preferred ligation orientation. For example, an otherwise untreated and undigested PCR product that has two blunt ends will typically not have a preferred ligation orientation when ligated into a cloning vector digested to produce blunt ends in its multiple cloning site; thus, directional ligation will typically not be displayed under these circumstances. In contrast, directional ligation will typically displayed when a digested PCR product having a 5' EcoR I-treated end and a 3' BamH I-is ligated into a cloning vector that has a multiple cloning site digested with EcoR I and BamH I.

SThe term "DNA shuffling" is used herein to indicate recombination between substantially homologous but non-identical sequences, in some embodiments DNA shuffling may involve crossover via non-homologous recombination, such as via cer/lox and/or flp/frt systems and the like.

00 As used in this invention, the term "epitope" refers to an antigenic r determinant on an antigen, such as a phytase polypeptide, to which the paratope of an antibody, such as an phytase-specific antibody, binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. As used herein "epitope" refers to that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding body of an antibody. Typically, such binding interaction is manifested as an intermolecular contact with one or more amino acid residues of a CDR.

The terms "fragment", "derivative" and "analog" when referring to a reference polypeptide comprise a polypeptide which retains at least one biological function or activity that is at least essentially same as that of the reference polypeptide. Furthermore, the terms "fragment", "derivative" or "analog" are exemplified by a "pro-form" molecule, such as a low activity proprotein that can be modified by cleavage to produce a mature enzyme with significantly higher activity.

A method is provided herein for producing from a template polypeptide a set of progeny polypeptides in which a "full range of single amino acid substitutions" is represented at each amino acid position. As used herein, "full range of single amino acid substitutions" is in reference to the naturally encoded 20 naturally encoded polypeptide-forming alpha-amino acids, as described herein.

The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region 0 (leader and trailer) as well as intervening sequences (introns) between individual C'i coding segments (exons).

"Genetic instability", as used herein, refers to the natural tendency of highly repetitive sequences to be lost through a process of reductive events generally O) involving sequence simplification through the loss of repeated sequences. Deletions tend to involve the loss of one copy of a repeat and everything between the repeats.

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t The term "heterologous" means that one single-stranded nucleic acid 010 sequence is unable to hybridize to another single-stranded nucleic acid sequence or C'l its complement. Thus areas of heterology means that areas ofpolynucleotides or polynucleotides have areas or regions within their sequence which are unable to hybridize to another nucleic acid or polynucleotide. Such regions or areas are for example areas of mutations.

The term "homologous" or "homeologous" means that one single-stranded nucleic acid nucleic acid sequence may hybridize to a complementary singlestranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentrations as discussed later. Preferably the region of identity is greater than about 5 bp, more preferably the region of identity is greater than 10 bp.

An immunoglobulin light or heavy chain variable region consists of a "framework" region interrupted by three hypervariable regions, also called CDR's.

The extent of the framework region and CDR's have been precisely defined; see "Sequences of Proteins of Immunological Interest" (Kabat et al, 1987). The sequences of the framework regions of different light or heavy chains are relatively conserved within a specie. As used herein, a "human framework region" is a framework region that is substantially identical (about 85 or more, usually 90-95 or more) to the framework region of a naturally occurring human immunoglobulin. the framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDR's. The SCDR's are primarily responsible for binding to an epitope of an antigen.

_The benefits of this invention extend to "industrial applications" (or industrial processes), which term is used to include applications in commercial OC industry proper (or simply industry) as well as non-commercial industrial applications biomedical research at a non-profit institution). Relevant applications include those in areas of diagnosis, medicine, agriculture, N manufacturing, and academia.

The term "identical" or "identity" means that two nucleic acid sequences have the same sequence or a complementary sequence. Thus, "areas of identity" means that regions or areas of a polynucleotide or the overall polynucleotide are identical or complementary to areas of another polynucleotide or the polynucleotide.

The term "isolated" means that the material is removed from its original environment the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated, but the same polynucleotide or enzyme, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

By "isolated nucleic acid" is meant a nucleic acid, a DNA or RNA molecule, that is not immediately contiguous with the 5' and 3' flanking sequences with which it normally is immediately contiguous when present in the naturally occurring genome of the organism from which it is derived. The term thus describes, for example, a nucleic acid that is incorporated into a vector, such as a plasmid or viral vector; a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell, but at a site different from that at which it naturally occurs); and a nucleic acid that exists as a separate O molecule, a DNA fragment produced by PCR amplification or restriction ,1 enzyme digestion, or an RNA molecule produced by in vitro transcription. The term Salso describes a recombinant nucleic acid that forms part of a hybrid gene encoding additional polypeptide sequences that can be used, for example, in the production of a fusion protein.

00 0As used herein "ligand" refers to a molecule, such as a random peptide or 0variable segment sequence, that is recognized by a particular receptor. As one of n skill in the art will recognize, a molecule (or macromolecular complex) can be both a receptor and a ligand. In general, the binding partner having a smaller molecular weight is referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor.

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Sambrook et al, 1982, p. 146; Sambrook, 1989). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units ofT4 DNA ligase ("ligase") per 0.5 tg of approximately equimolar amounts of the DNA fragments to be ligated.

As used herein, "linker" or "spacer" refers to a molecule or group of molecules that connects two molecules, such as a DNA binding protein and a random peptide, and serves to place the two molecules in a preferred configuration, so that the random peptide can bind to a receptor with minimal steric hindrance from the DNA binding protein.

As used herein, a "molecular property to be evolved" includes reference to molecules comprised of a polynucleotide sequence, molecules comprised of a polypeptide sequence, and molecules comprised in part of a polynucleotide sequence and in part of a polypeptide sequence. Particularly relevant but by no means limiting examples of molecular properties to be evolved include enzymatic activities at specified conditions, such as related to temperature; salinity; pressure; pH; and concentration of glycerol, DMSO, detergent, &/or any other molecular 0 species with which contact is made in a reaction environment. Additional

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N particularly relevant but by no means limiting examples of molecular properties Sto be evolved include stabilities e.g. the amount of a residual molecular property that is present after a specified exposure time to a specified environment, such as may be encountered during storage.

00 The term "mutations" means changes in the sequence of a wild-type nucleic Sacid sequence or changes in the sequence of a peptide. Such mutations may be point t mutations such as transitions or transversions. The mutations may be deletions, insertions or duplications.

As used herein, the degenerate nucleotide sequence represents 32 possible triplets, where can be A, C, G or T.

The term "naturally-occurring" as used herein as applied to the object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. Generally, the term naturally occurring refers to an object as present in a non-pathological (un-diseased) individual, such as would be typical for the species.

As used herein, a "nucleic acid molecule" is comprised of at least one base or one base pair, depending on whether it is single-stranded or double-stranded, respectively. Furthermore, a nucleic acid molecule may belong exclusively or chimerically to any group ofnucleotide-containing molecules, as exemplified by, but not limited to, the following groups of nucleic acid molecules: RNA, DNA, genomic nucleic acids, non-genomic nucleic acids, naturally occurring and not naturally occurring nucleic acids, and synthetic nucleic acids. This includes, by way of non-limiting example, nucleic acids associated with any organelle, such as the mitochondria, ribosomal RNA, and nucleic acid molecules comprised chimerically 0 of one or more components that are not naturally occurring along with naturally N occurring components.

Additionally, a "nucleic acid molecule" may contain in part one or more non-nucleotide-based components as exemplified by, but not limited to, amino acids 00 and sugars. Thus, by way of example, but not limitation, a ribozyme that is in part 0 nucleotide-based and in part protein-based is considered a "nucleic acid molecule".

t In addition, by way of example, but not limitation, a nucleic acid molecule that is labeled with a detectable moiety, such as a radioactive or alternatively a nonradioactive label, is likewise considered a "nucleic acid molecule".

The terms "nucleic acid sequence coding for" or a "DNA coding sequence of" or a "nucleotide sequence encoding" a particular enzyme as well as other synonymous terms refer to a DNA sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulatory sequences.

A "promotor sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. The promoter is part of the DNA sequence. This sequence region has a start codon at its 3' terminus. The promoter sequence does include the minimum number of bases where elements necessary to initiate transcription at levels detectable above background. However, after the RNA polymerase binds the sequence and transcription is initiated at the start codon terminus with a promoter), transcription proceeds downstream in the 3' direction. Within the promotor sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1) as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.

The terms "nucleic acid encoding an enzyme (protein)" or "DNA encoding an enzyme (protein)" or "polynucleotide encoding an enzyme (protein)" and other synonymous terms encompasses a polynucleotide which includes only coding O sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.

In one preferred embodiment, a "specific nucleic acid molecule species" is defined by its chemical structure, as exemplified by, but not limited to, its primary 00oO sequence. In another preferred embodiment, a specific "nucleic acid molecule species" is defined by a function of the nucleic acid species or by a function of a product derived from the nucleic acid species. Thus, by way of non-limiting example, a "specific nucleic acid molecule species" may be defined by one or more activities or properties attributable to it, including activities or properties attributable its expressed product.

The instant definition of "assembling a working nucleic acid sample into a nucleic acid library" includes the process of incorporating a nucleic acid sample into a vector-based collection, such as by ligation into a vector and transformation of a host. A description of relevant vectors, hosts, and other reagents as well asspecific non-limiting examples thereof are provided hereinafter. The instant definition of "assembling a working nucleic acid sample into a nucleic acid library" also includes the process of incorporating a nucleic acid sample into a nonvector-based collection, such as by ligation to adaptors. Preferably the adaptors can anneal to PCR primers to facilitate amplification by PCR.

Accordingly, in a non-limiting embodiment, a "nucleic acid library" is comprised of a vector-based collection of one or more nucleic acid molecules. In another preferred embodiment a "nucleic acid library" is comprised of a nonvector-based collection of nucleic acid molecules. In yet another preferred embodiment a "nucleic acid library" is comprised of a combined collection of nucleic acid molecules that is in part vector-based and in part non-vector-based.

Preferably, the collection of molecules comprising a library is searchable and separable according to individual nucleic acid molecule species.

0 The present invention provides a "nucleic acid construct" or alternatively a

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Ci "nucleotide construct" or alternatively a "DNA construct". The term "construct" Sis used herein to describe a molecule, such as a polynucleotide a phytase polynucleotide) may optionally be chemically bonded to one or more additional molecular moieties, such as a vector, or parts of a vector. In a specific but by no OC means limiting aspect, a nucleotide construct is exemplified by a DNA expression DNA expression constructs suitable for the transformation of a host cell.

ci it An "oligonucleotide" (or synonymously an "oligo") refers to either a single 0 10 stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides may or may not have a 5' phosphate. Those that do not will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. To achieve polymerase-based amplification (such as with PCR), a "32-fold degenerate oligonucleotide that is comprised of, in series, at least a first homologous sequence, a degenerate N,N,G/T sequence, and a second homologous sequence" is mentioned. As used in this context, "homologous" is in reference to homology between the oligo and the parental polynucleotide that is subjected to the polymerase-based amplification.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

A coding sequence is "operably linked to" another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from 0 both coding sequences. The coding sequences need not be contiguous to one CN another so long as the expressed sequences are ultimately processed to produce the Sdesired protein.

As used herein the term "parental polynucleotide set" is a set comprised of 0 one or more distinct polynucleotide species. Usually this term fis used in reference to a progeny polynucleotide set which is preferably obtained by mutagenization of Sthe parental set, in which case the terms "parental", "starting" and "template" are t used interchangeably.

As used herein the term "physiological conditions" refers to temperature, pH, ionic strength, viscosity, and like biochemical parameters which are compatible with a viable organism, and/or which typically exist intracellularly in a viable cultured yeast cell or mammalian cell. For example, the intracellular conditions in a yeast cell grown under typical laboratory culture conditions are physiological conditions. Suitable in vitro reaction conditions for in vitro transcription cocktails are generally physiological conditions. In general, in vitro physiological conditions comprise 50-200 mM NaCl or KC1, pH 6.5-8.5, 20-45 0 C and 0.001-10 mM divalent cation Mg", preferably about 150 mM NaCl or KC1, pH 7.2-7.6, 5 mM divalent cation, and often include 0.01-1.0 percent nonspecific protein BSA).

A non-ionic detergent (Tween, NP-40, Triton X-100) can often be present, usually at about 0.001 to typically 0.05-0.2% Particular aqueous conditions may be selected by the practitioner according to conventional methods. For general guidance, the following buffered aqueous conditions may be applicable: 10-250 mM NaCI, 5-50 mM Tris HCI, pH 5-8, with optional addition of divalent cation(s) and/or metal chelators and/or non-ionic detergents and/or membrane fractions and/or anti-foam agents and/or scintillants.

Standard convention to is used herein to describe the sequence of double standed polynucleotides.

0 The term "population" as used herein means a collection of components ,1 such as polynucleotides, portions or polynucleotides or proteins. A "mixed Spopulation: means a collection of components which belong to the same family of nucleic acids or proteins are related) but which differ in their sequence are not identical) and hence in their biological activity.

00 0A molecule having a "pro-form" refers to a molecule that undergoes any Scombination of one or more covalent and noncovalent chemical modifications (e.g.

tt) glycosylation, proteolytic cleavage, dimerization or oligomerization, temperatureinduced or pH-induced conformational change, association with a co-factor, etc.) en route to attain a more mature molecular form having a property difference an increase in activity) in comparison with the reference pro-form molecule. When two or more chemical modification two proteolytic cleavages, or a proteolytic cleavage and a deglycosylation) can be distinguished en route to the production of a mature molecule, the referemce precursor molecule may be termed a "pre-proform" molecule.

As used herein, the term "pseudorandom" refers to a set of sequences that have limited variability, such that, for example, the degree of residue variability at another position, but any pseudorandom position is allowed some degree of residue variation, however circumscribed.

"Quasi-repeated units", as used herein, refers to the repeats to be reassorted and are by definition not identical. Indeed the method is proposed not only for practically identical encoding units produced by mutagenesis of the identical starting sequence, but also the reassortment of similar or related sequences which may diverge significantly in some regions. Nevertheless, if the sequences contain sufficient homologies to be reassorted by this approach, they can be referred to as "quasi-repeated" units.

As used herein "random peptide library" refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and to the set of random peptides 0 encoded by those polynucleotide sequences, as well as the fusion proteins contain

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N those random peptides.

As used herein, "random peptide sequence" refers to an amino acid sequence composed of two or more amino acid monomers and constructed by a o stochastic or random process. A random peptide can include framework or scaffolding motifs, which may comprise invariant sequences.

SAs used herein, "receptor" refers to a molecule that has an affinity for a 0 10 given ligand. Receptors can be naturally occurring or synthetic molecules.

Receptors can be employed in an unaltered state or as aggregates with other species.

Receptors can be attached, covalently or non-covalently, to a binding member, either directly or via a specific binding substance. Examples of receptors include, but are not limited to, antibodies, including monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells, or other materials), cell membrane receptors, complex carbohydrates and glycoproteins, enzymes, and hormone receptors.

"Recombinant" enzymes refer to enzymes produced by recombinant DNA techniques, produced from cells transformed by an exogenous DNA construct encoding the desired enzyme. "Synthetic" enzymes are those prepared by chemical synthesis.

The term "related polynucleotides" means that regions or areas of the polynucleotides are identical and regions or areas of the polynucleotides are heterologous.

"Reductive reassortment", as used herein, refers to the increase in molecular diversity that is accrued through deletion (and/or insertion) events that are mediated by repeated sequences.

0 The following terms are used to describe the sequence relationships between

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CN two or more polynucleotides: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." 00 A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, or Smay comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides and may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.

"Repetitive Index as used herein, is the average number of copies of the quasi-repeated units contained in the cloning vector.

The term "restriction site" refers to a recognition sequence that is necessary for the manifestation of the action of a restriction enzyme, and includes a site of catalytic cleavage. It is appreciated that a site of cleavage may or may not be contained within a portion of a restriction site that comprises a low ambiguity sequence a sequence containing the principal determinant of the frequency of occurrence of the restriction site). Thus, in many cases, relevant restriction sites contain only a low ambiguity sequence with an internal cleavage site (e.g.

G/AATTC in the EcoR I site) or an immediately adjacent cleavage site (e.g.

/CCWGG in the EcoR II site). In other cases, relevant restriction enzymes the Eco57 I site or CTGAAG(16/14)] contain a low ambiguity sequence the CTGAAG sequence in the Eco57 I site) with an external cleavage site in the 6 N 1 6 portion of the Eco57 I site). When an enzyme a restriction enzyme) is said

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Ci to "cleave" a polynucleotide, it is understood to mean that the restriction enzyme catalyzes or facilitates a cleavage of a polynucleotide.

In a non-limiting aspect, a "selectable polynucleotide" is comprised of a oo terminal region (or end region), an intermediate region an internal or central region), and a 3' terminal region (or end region). As used in this aspect, a Sterminal region is a region that is located towards a 5' polynucleotide terminus (or a polynucleotide end); thus it is either partially or entirely in a 5' half of a polynucleotide. Likewise, a 3' terminal region is a region that is located towards a 3' polynucleotide terminus (or a 3' polynucleotide end); thus it is either partially or entirely in a 3' half of a polynucleotide. As used in this non-limiting exemplification, there may be sequence overlap between any two regions or even among all three regions.

The term "sequence identity" means that two polynucleotide sequences are identical on a nucleotide-by-nucleotide basis) over the window of comparison.

The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison the window size), and multiplying the result by 100 to yield the percentage of sequence identity. This "substantial identity", as used herein, denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 80 percent sequence identity, preferably at least percent identity, often 90 to 95 percent sequence identity, and most commonly at least 99 percent sequence identity as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total percent or less of the reference sequence over the window of comparison.

As known in the art "similarity" between two enzymes is determined by Scomparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of a second enzyme. Similarity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic 00 Local Alignment Search Tool at the National Center for Biological Information).

OAs used herein, the term "single-chain antibody" refers to a polypeptide t comprising a VH domain and a VL domain in polypeptide linkage, generally liked via a spacer peptide [Gly-Gly-Gly-Gly-Ser]x), and which may comprise additional amino acid sequences at the amino- and/or carboxy- termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a scFv is a single-chain antibody. Singlechain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino substantially encoded by genes of the immunoglobulin superfamily see Williams and Barclay, 1989, pp. 361-368, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine bovine, ovine, goat, or human heavy chain or light chain gene sequence. A functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).

The members of a pair of molecules an antibody-antigen pair or a nucleic acid pair) are said to "specifically bind" to each other if they bind to each other with greater affinity than to other, non-specific molecules. For example, an antibody raised against an antigen to which it binds more efficiently than to a nonspecific protein can be described as specifically binding to the antigen. (Similarly, a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions (see above).) 0 "Specific hybridization" is defined herein as the formation of hybrids 0 Ci between a first polynucleotide and a second polynucleotide a polynucleotide having a distinct but substantially identical sequence to the first polynucleotide), wherein substantially unrelated polynucleotide sequences do not form hybrids in the mixture.

00 The term "specific polynucleotide" means a polynucleotide having certain end points and having a certain nucleic acid sequence. Two polynucleotides wherein t one polynucleotide has the identical sequence as a portion of the second polynucleotide but different ends comprises two different specific polynucleotides.

"Stringent hybridization conditions" means hybridization will occur only if there is at least 90% identity, preferably at least 95% identity and most preferably at least 97% identity between the sequences. See Sambrook et al, 1989, which is hereby incorporated by reference in its entirety.

Also included in the invention are polypeptides having sequences that are "substantially identical" to the sequence of a phytase polypeptide, such as one of SEQ ID 1. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence only by conservative amino acid substitutions, for example, substitutions of one amino acid for another of the same class substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution ofarginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine).

Additionally a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence or by one or more non-conservative substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site the molecule, and provided that the polypeptide essentially retains its behavioural properties. For example, one or more amino acids can be deleted from a phytase polypeptide, resulting in modification of the structure

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of the polypeptide, without significantly altering its biological activity. For C example, amino- or carboxyl-terminal amino acids that are not required for phytase Sbiological activity can be removed. Such modifications can result in the development of smaller active phytase polypeptides.

00 The present invention provides a "substantially pure enzyme". The term S"substantially pure enzyme" is used herein to describe a molecule, such as a Spolypeptide a phytase polypeptide, or a fragment thereof) that is substantially Sfree of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated. For example, a substantially pure molecule, such as a polypeptide, can be at least 60%, by dry weight, the molecule of interest. The purity of the polypeptides can be determined using standard methods including, polyacrylamide gel electrophoresis SDS-PAGE), column chromatography high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis.

As used herein, "substantially pure" means an object species is the predominant species present on a molar basis it is more abundant than any other individual macromolecular species in the composition), and preferably substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

Generally, a substantially pure composition will comprise more than about 80 to percent of all macromolecular species present in the composition. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species.

As used herein, the term "variable segment" refers to a portion of a nascent peptide which comprises a random, pseudorandom, or defined kernal sequence. A variable segment" refers to a portion of a nascent peptide which comprises a random Spseudorandom, or defined kemrnal sequence. A variable segment can comprise both C variant and invariant residue positions, and the degree of residue variation at a Zvariant residue position may be limited: both options are selected at the discretion of the practitioner. Typically, variable segments are about 5 to 20 amino acid residues in length 8 to 10), although variable segments may be longer and may O comprise antibody portions or receptor proteins, such as an antibody fragment, a nucleic acid binding protein, a receptor protein, and the like.

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SThe term "wild-type" means that the polynucleotide does not comprise any mutations. A "wild type" protein means that the protein will be active at a level of activity found in nature and will comprise the amino acid sequence found in nature.

The term "working", as in "working sample", for example, is simply a sample with which one is working. Likewise, a '"working molecule", for example is a molecule with which one is working.

DETAILED DESCRIPTION OF THE INVENTION The invention described herein is directed to the use of repeated cycles of reductive reassortment, recombination and selection which allow for the directed molecular evolution of highly complex linear sequences, such as DNA, RNA or 00 proteins thorough recombination.

0 0In vivo shuffling of molecules can be performed utilizing the natural property t of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.

In a preferred embodiment, the invention relates to a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide. The present invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell. The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide. The term "hybrid polynucleotide", as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.

0 The invention provides a means for generating hybrid polynucleotides which 0 CN may encode biologically active hybrid polypeptides. In one aspect, the original Spolynucleotides encode biologically active polypeptides. The method of the invention produces new hybrid polypeptides by utilizing cellular processes which integrate the sequence of the original polynucleotides such that the resulting hybrid oo polynucleotide encodes a polypeptide demonstrating activities derived from the Soriginal biologically active polypeptides. For example, the original polynucleotides may encode a particular enzyme from different microorganisms. An enzyme Sencoded by a first polynucleotide from one organism may, for example, function 010 effectively under a particular environmental condition, e.g. high salinity. An enzyme encoded by a second polynucleotide from a different organism may function effectively under a different environmental condition, such as extremely high temperatures. A hybrid polynucleotide c6ntaining sequences from the first and second original polynucleotides may encode an enzyme which exhibits characteristics of both enzymes encoded by the original polynucleotides. Thus, the enzyme encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the enzymes encoded by the first and second polynucleotides, high salinity and extreme temperatures.

Enzymes encoded by the original polynucleotides of the invention include, but are not limited to; oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. A hybrid polypeptide resulting from the method of the invention may exhibit specialized enzyme activity not displayed in the original enzymes. For example, following recombination and/or reductive reassortment of polynucleotides encoding hydrolase activities, the resulting hybrid polypeptide encoded by a hybrid polynucleotide can be screened for specialized hydrolase activities obtained from each of the original enzymes, i.e. the type of bond on which the hydrolase acts and the temperature at which the hydrolase functions. Thus, for example, the hydrolase may be screened to ascertain those chemical functionalities which distinguish the hybrid hydrolase from the original hydrolyases, such as: amide (peptide bonds), i.e. proteases; ester bonds, i.e. esterases and lipases; acetals, glycosidases O and, for example, the temperature, pH or salt concentration at which the hybrid C polypeptide functions.

Sources of the original polynucleotides may be isolated from individual organisms ("isolates"), collections of organisms that have been grown in defined oO media ("enrichment cultures"), or, most preferably, uncultivated organisms 0 ("environmental samples"). The use of a culture-independent approach to derive 0polynucleotides encoding novel bioactivities from environmental samples is most preferable since it allows one to access untapped resources of biodiversity.

O "Environmental libraries" are generated from environmental samples and represent the collective genomes of naturally occurring organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction ofprokaryotes that can be grown in pure culture.

Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample. This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample which may be under-represented by several orders of magnitude compared to the dominant species.

For example, gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest. Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries. Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell. The host cell is grown under conditions which promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities.

The microorganisms from which the polynucleotide may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae and protozoa. Polynucleotides Q may be isolated from environmental samples in which case the nucleic acid may be Srecovered without the culturing of an organism or recovered from one or more cultured organisms. In one aspect, such microorganisms may be extremophiles, such _as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles. Polynucleotides encoding enzymes isolated from extremophilic microorganisms are particularly preferred. Such enzymes may function at temperatures above 100 0 C in terrestrial hot springs and deep sea thermal vents, at c temperatures below 0°C in arctic waters, in the saturated salt environment of the SDead Sea, at pH values around 0 in coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in sewage sludge. For example, several esterases and C lipases cloned and expressed from extremophilic organisms show high activity throughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell. A suitable host cell is any cell which is capable of promoting recombination and/or reductive reassortment. The selected polynucleotides are preferably already in a vector which includes appropriate control sequences. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al, 1986).

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

With particular references to various mammalian cell culture systems that can be employed to express recombinant protein, examples of mammalian 6 expression systems include the COS-7 lines of monkey kidney fibroblasts, described

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C in "SV40-transformed simian cells support the replication of early SV40 mutants" (Gluzman, 1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and o also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites Smay be used to provide the required nontranscribed genetic elements.

Host cells containing the polynucleotides of interest can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the enhanced activity.

In another aspect, it is envisioned the method of the present invention can be used to generate novel polynucleotides encoding biochemical pathways from one or more operons or gene clusters or portions thereof. For example, bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes. The genes are clustered, in structures referred to as "gene clusters," on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster. Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function. An example of a biochemical pathway encoded by gene clusters are polyketides. Polyketides are molecules which are an extremely rich source ofbioactivities, including antibiotics (such as tetracyclines and erythromycin), anti-cancer agents (daunomycin), immunosuppressants (FK506 and rapamycin), and veterinary products (monensin).

0 Many polyketides (produced by polyketide synthases) are valuable as therapeutic CN agents. Polyketide synthases are multifunctional enzymes that catalyze the biosynthesis of an enormous variety of carbon chains differing in length and patterns of functionality and cyclization. Polyketide synthase genes fall into gene clusters and at least one type (designated type I) of polyketide synthases have large size 00 genes and enzymes, complicating genetic manipulation and in vitro studies of these genes/proteins.

t) The ability to select and combine desired components from a library of 0 10 polyketides, or fragments thereof, and postpolyketide biosynthesis genes for generation of novel polyketides for study is appealing. The method of the present invention makes it possible to facilitate the production of novel polyketide synthases through intermolecular recombination.

Preferably, gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or proteinrelated array activity from the ligated gene clusters. Use of vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E. coli. This f-factor ofE. coli is a plasmid which affect high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples. Once ligated into an appropriate vector, two or more vectors containing different polyketide synthase gene clusters can be introduced into a suitable host cell. Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster. The novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.

0 Therefore, in a preferred embodiment, the present invention relates to a C method for producing a biologically active hybrid polypeptide and screening such a Spolypeptide for enhanced activity by: 1) introducing at least a first polynucleotide in operable linkage and a second polynucleotide in operable linkage, said at least first 00 polynucleotide and second polynucleotide sharing at least one region of 0 partial sequence homology, into a suitable host cell; 2) growing the host cell under conditions which promote sequence in reorganization resulting in a hybrid polynucleotide in operable linkage; O 10 3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide; 4) screening the hybrid polypeptide under conditions which promote identification of enhanced biological activity; and isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various enzyme activities are known to those of skill in the art and discussed throughout the present specification. Such methods may be employed when isolating the polypeptides and polynucleotides of the present invention.

As representative examples of expression vectors which may be used there may be mentioned viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pi-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, aspergillus and yeast).

Thus, for example, the DNA may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXTI, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or Sother vector may be used as long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present _invention.

0A preferred type of vector for use in the present invention contains an f-factor origin replication. The f-factor (or fertility factor) in E. coli is a plasmid which effects high frequency transfer of itself during conjugation and less frequent transfer of the bacterial chromosome itself. A particularly preferred embodiment is to use cloning vectors, referred to as "fosmids" or bacterial artificial chromosome c (BAC) vectors. These are derived from E. coli f-factor which is able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable "environmental DNA library." Another preferred type of vector for use in the present invention is a cosmid vector. Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA. Cloning into cosmid vectors is described in detail in "Molecular Cloning: A laboratory Manual" (Sambrook et al, 1989).

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct RNA synthesis.

Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.

O In addition, the expression vectors preferably contain one or more selectable N marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

0 Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, the ampicillin Sresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from t a highly-expressed gene to direct transcription of a downstream structural sequence.

Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.

The cloning strategy permits expression via both vector driven and endogenous promoters; vector promotion may be important with expression of genes whose endogenous promoter will not function in E. coli.

The DNA isolated or derived from microorganisms can preferably be inserted into a vector or a plasmid prior to probing for selected DNA. Such vectors or plasmids are preferably those containing expression regulatory sequences, including promoters, enhancers and the like. Such polynucleotides can be part of a vector and/or a composition and still be isolated, in that such vector or composition is not part of its natural environment. Particularly preferred phage or plasmid and methods for introduction and packaging into them are described in detail in the protocol set forth herein.

The selection of the cloning vector depends upon the approach taken, for example, the vector can be any cloning vector with an adequate capacity to multiply repeated copies of a sequence, or multiple sequences that can be successfully O transformed and selected in a host cell. One example of such a vector is described in CN "Polycos vectors: a system for packaging filamentous phage and phagemid vectors Susing lambda phage packaging extracts" (Alting-Mecs and Short, 1993).

Propagation/maintenance can be by an antibiotic resistance carried by the cloning vector. After a period of growth, the naturally abbreviated molecules are recovered 00 and identified by size fractionation on a gel or column, or amplified directly. The Scloning vector utilized may contain a selectable gene that is disrupted by the insertion of the lengthy construct. As reductive reassortment progresses, the number t of repeated units is reduced and the interrupted gene is again expressed and hence selection for the processed construct can be applied. The vector may be an expression/selection vector which will allow for the selection of an expressed product possessing desirable biologically properties. The insert may be positioned downstream of a functional promotor and the desirable property screened by appropriate means.

In vivo reassortment is focused on "inter-molecular" processes collectively referred to as "recombination" which in bacteria, is generally viewed as a "RecAdependent" phenomenon. The present invention can rely on recombination processes of a host cell to recombine and re-assort sequences, or the cells' ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion. This process of "reductive reassortment" occurs by an "intramolecular", RecA-independent process.

Therefore, in another aspect of the present invention, novel polynucleotides can be generated by the process of reductive reassortment. The method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropriate host cell. The reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasirepeated units. The reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel b molecular species. Various treatments may be applied to enhance the rate of N, reassortment. These could include treatment with ultra-violet light, or DNA damaging chemicals, and/or the use of host cell lines displaying enhanced levels of "genetic instability". Thus the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their 00 own evolution.

SRepeated or "quasi-repeated" sequences play a role in genetic instability. In Sthe present invention, "quasi-repeats" are repeats that are not restricted to their original unit structure. Quasi-repeated units can be presented as an array of sequences in a construct; consecutive units of similar sequences. Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level. The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi-repeated sequences. The quasirepeated units provide a practically limitless repertoire of templates upon which slippage events can occur. The constructs containing the quasi-repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units.

When the quasi-repeated sequences are all ligated in the same orientation, for instance head to tail or vice versa, the cell cannot distinguish individual units.

Consequently, the reductive process can occur throughout the sequences. In contrast, when for example, the units are presented head to head, rather than head to tail, the inversion delineates the endpoints of the adjacent unit so that deletion formation will favor the loss of discrete units. Thus, it is preferable with the present method that the sequences are in the same orientation. Random orientation of quasirepeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequences will offer the highest efficiency. However, while having fewer of the contiguous sequences in the same orientation decreases the efficiency, it may still provide sufficient elasticity for the effective recovery of novel molecules.

b Constructs can be made with the quasi-repeated sequences in the same orientation to callow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following: 00 a) Primers that include a poly-A head and poly-T tail which when made single-stranded would provide orientation can be utilized. This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNAseH.

b) Primers that include unique restriction cleavage sites can be utilized.

Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required.

c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identification of cloning vectors with a reduced RI. The re-assorted encoding sequences can then be recovered by amplification. The products are re-cloned and expressed. The recovery of cloning vectors with reduced RI can be effected by: 1) The use of vectors only stably maintained when the construct is reduced in complexity.

2) The physical recovery of shortened vectors by physical procedures. In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on either an agarose gel, or column with a low molecular weight cut off utilizing standard procedures.

3) The recovery of vectors containing interrupted genes which can be selected when insert size decreases.

4) The use of direct selection techniques with an expression vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms may demonstrate a high degree of homology and encode quite diverse protein products.

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These types of sequences are particularly useful in the present invention as quasirepeats. However, while the examples illustrated below demonstrate the reassortment of nearly identical original encoding sequences (quasi-repeats), this process is not limited to such nearly identical repeats.

00 The following example demonstrates the method of the invention. Encoding nucleic acid sequences (quasi-repeats) derived from three unique species are depicted. Each sequence encodes a protein with a distinct set of properties. Each of F the sequences differs by a single or a few base pairs at a unique position in the I 10 sequence which are designated and The quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules. The number of quasi-repeat units can be controlled by the assembly conditions. The average number of quasi-repeated units in a construct is defined as the repetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell. The cells are then propagated and "reductive reassortment" is effected. The rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired. Whether the reduction in RI is mediated by deletion formation between repeated sequences by an "intra-molecular" mechanism, or mediated by recombination-like events through "inter-molecular" mechanisms is immaterial. The end result is a reassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact such as catalytic antibodies) with a predetermined macromolecule, such as for example a proteinaceous receptor, peptide oligosaccharide, viron, or other predetermined compound or structure.

Q The displayed polypeptides, antibodies, peptidomimetic antibodies, and variable region sequences that are identified from such libraries can be used for therapeutic, diagnostic, research and related purposes catalysts, solutes for increasing osmolarity of an aqueous solution, and the like), and/or can be subjected to one or more additional cycles of shuffling and/or affinity selection. The method 00 can be modified such that the step of selecting for a phenotypic characteristic can be

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Cc other than of binding affinity for a predetermined molecule for catalytic CI activity, stability oxidation resistance, drug resistance, or detectable phenotype 0conferred upon a host cell).

NC The present invention provides a method for generating libraries of displayed antibodies suitable for affinity interactions screening. The method comprises (1) obtaining first a plurality of selected library members comprising a displayed antibody and an associated polynucleotide encoding said displayed antibody, and obtaining said associated polynucleotide encoding for said displayed antibody and obtaining said associated polynucleotides or copies thereof, wherein said associated polynucleotides comprise a region of substantially identical variable region framework sequence, and introducing said polynucleotides into a suitable host cell and growing the cells under conditions which promote recombination and reductive reassortment resulting in shuffled polynucleotides. CDR combinations comprised by the shuffled pool are not present in the first plurality of selected library members, said shuffled pool composing a library of displayed antibodies comprising CDR permutations and suitable for affinity interaction screening.

Optionally, the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope (antigen) and thereby selecting a plurality of selected shuffled library members. Further, the plurality of selectively shuffled library members can be shuffled and screened iteratively, from 1 to about 1000 cycles or as desired until library members having a desired binding affinity are obtained.

In another aspect of the invention, it is envisioned that prior to or during recombination or reassortment, polynucleotides generated by the method of the present invention can be subjected to agents or processes which promote the introduction of mutations into the original polynucleotides. The introduction of such mutations would increase the diversity of resulting hybrid polynucleotides and polypeptides encoded therefrom. The agents or processes which promote mutagenesis can include, but are not limited to: (+)-CC-1065, or a synthetic analog 00 Ssuch as (+)-CC-1065-(N3-Adenine, see Sun and Hurley, 1992); an N-acelylated or C deacetylated 4'-fluro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis C' (see, for example, van de Poll et al, 1992); or a N-acetylated or deacetylated 4- Saminobiphenyl adduct capable of inhibiting DNA synthesis (see also, van de Poll et C' 10 al, 1992, pp. 751-758); trivalent chromium, a trivalent chromium salt, a polycyclic aromatic hydrocarbon DNA adduct capable of inhibiting DNA replication, such as 7 -bromomethyl-benz[a]anthracene tris(2,3dibromopropyl)phosphate ("Tris-BP"), 1,2-dibromo-3-chloropropane 2bromoacrolein (2BA), benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide a platinum(II) halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-j-quinoline ("N-hydroxy-IQ"), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine ("N-hydroxy-PhIP"). Especially preferred "means for slowing or halting PCR amplification consist of UV light (+)-CC-1065 and (+)-CC-1065-(N3- Adenine). Particularly encompassed means are DNA adducts or polynucleotides comprising the DNA adducts from the polynucleotides or polynucleotides pool, which can be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing.

In another aspect the present invention is directed to a method of producing recombinant proteins having biological activity by treating a sample comprising double-stranded template polynucleotides encoding a wild-type protein under conditions according to the present invention which provide for the production of hybrid or re-assorted polynucleotides.

The invention also provides the use of polynucleotide shuffling to shuffle a population of viral genes capsid proteins, spike glycoproteins, polymerases, and proteases) or viral genomes paramyxoviridae, orthomyxoviridae, Sherpesviruses, retroviruses, reoviruses and rhinoviruses). In an embodiment, the invention provides a method for shuffling sequences encoding all or portions of immunogenic viral proteins to generate novel combinations of epitopes as well as novel epitopes created by recombination; such shuffled viral proteins may comprise epitopes or combinations of epitopes as well as novel epitopes created by oO Srecombination; such shuffled viral proteins may comprise epitopes or combinations

O

Sof epitopes which are likely to arise in the natural environment as a consequence of C viral evolution; such as recombination of influenza virus strains).

N 10 The invention also provides a method suitable for shuffling polynucleotide sequences for generating gene therapy vectors and replication-defective gene therapy constructs, such as may be used for human gene therapy, including but not limited to vaccination vectors for DNA-based vaccination, as well as anti-neoplastic gene therapy and other general therapy formats.

In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences".

Methodology Nucleic acid shuffling is a method for in vitro orin vivo homologous recombination of pools of shorter or smaller polynucleotides to produce a polynucleotide or polynucleotides. Mixtures of related nucleic acid sequences or polynucleotides are subjected to sexual PCR to provide random polynucleotides, and 0 reassembled to yield a library or mixed population of recombinant hybrid nucleic Sacid molecules or polynucleotides.

In contrast to cassette mutagenesis, only shuffling and error-prone PCR allow one to mutate a pool of sequences blindly (without sequence information other OC than primers).

SThe advantage of the mutagenic shuffling of this invention over error-prone PCR alone for repeated selection can best be explained with an example from 1 10 antibody engineering. Consider DNA shuffling as compared with error-prone PCR (not sexual PCR). The initial library of selected pooled sequences can consist of related sequences of diverse origin antibodies from naive mRNA) or can be derived by any type of mutagenesis (including shuffling) of a single antibody gene.

A collection of selected complementarity determining regions ("CDRs") is obtained after the first round of affinity selection. In the diagram the thick CDRs confer onto the antibody molecule increased affinity for the antigen. Shuffling allows the free combinatorial association of all of the CDRls with all of the CDR2s with all of the CDR3s, for example.

This method differs from error-prone PCR, in that it is an inverse chain reaction. In error-prone PCR, the number ofpolymerase start sites and the number of molecules grows exponentially. However, the sequence of the polymerase start sites and the sequence of the molecules remains essentially the same. In contrast, in nucleic acid reassembly or shuffling of random polynucleotides the number of start sites and the number (but not size) of the random polynucleotides decreases over time. For polynucleotides derived from whole plasmids the theoretical endpoint is a single, large concatemeric molecule.

Since cross-overs occur at regions of homology, recombination will primarily occur between members of the same sequence family. This discourages combinations of CDRs that are grossly incompatible directed against different epitopes of the same antigen). It is contemplated that multiple families of sequences 0 can be shuffled in the same reaction. Further, shuffling generally conserves the relative order, such that, for example, CDRI will not be found in the position of CDR2.

Rare shufflants will contain a large number of the best (eg. highest affinity) oO CDRs and these rare shufflants may be selected based on their superior affinity.

O

C1 CDRs from a pool of 100 different selected antibody sequences can be O permutated in up to 1006 different ways. This large number of permutations cannot N 10 be represented in a single library of DNA sequences. Accordingly, it is contemplated that multiple cycles of DNA shuffling and selection may be required depending on the length of the sequence and the sequence diversity desired.

Error-prone PCR, in contrast, keeps all the selected CDRs in the same relative sequence, generating a much smaller mutant cloud.

The template polynucleotide which may be used in the methods of this invention may be DNA or RNA. It may be of various lengths depending on the size of the gene or shorter or smaller polynucleotide to be recombined or reassembled.

Preferably, the template polynucleotide is from 50 bp to 50 kb. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest can be used in the methods of this invention, and in fact have been successfully used.

The template polynucleotide may be obtained by amplification using the PCR reaction (USPN 4,683,202 and USPN 4,683,195) or other amplification or cloning methods. However, the removal of free primers from the PCR products before subjecting them to pooling of the PCR products and sexual PCR may provide more efficient results. Failure to adequately remove the primers from the original pool before sexual PCR can lead to a low frequency of crossover clones.

The template polynucleotide often should be double-stranded. A double-stranded nucleic acid molecule is recommended to ensure that regions of the resulting single-stranded polynucleotides are complementary to each other and thus Scan hybridize to form a double-stranded molecule.

It is contemplated that single-stranded or double-stranded nucleic acid polynucleotides having regions of identity to the template polynucleotide and 00 Sregions of heterology to the template polynucleotide may be added to the template

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2C polynucleotide, at this step. It is also contemplated that two different but related C polynucleotide templates can be mixed at this step.

1 10 The double-stranded polynucleotide template and any added double-or single-stranded polynucleotides are subjected to sexual PCR which includes slowing or halting to provide a mixture of from about 5 bp to 5 kb or more. Preferably the size of the random polynucleotides is from about 10 bp to 1000 bp, more preferably the size of the polynucleotides is from about 20 bp to 500 bp.

Alternatively, it is also contemplated that double-stranded nucleic acid having multiple nicks may be used in the methods of this invention. A nick is a break in one strand of the double-stranded nucleic acid. The distance between such nicks is preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp. This can provide areas of self-priming to produce shorter or smaller polynucleotides to be included with the polynucleotides resulting from random primers, for example.

The concentration of any one specific polynucleotide will not be greater than 1% by weight of the total polynucleotides, more preferably the concentration of any one specific nucleic acid sequence will not be greater than 0.1% by weight of the total nucleic acid.

The number of different specific polynucletides in the mixture will be at least about 100, preferably at least about 500, and more preferably at least about 1000.

At this step single-stranded or double-stranded polynucleotides, either synthetic or natural, may be added to the random double-stranded shorter or smaller polynucleotides in order to increase the heterogeneity of the mixture of polynucleotides.

It is also contemplated that populations of double-stranded randomly broken polynucleotides may be mixed or combined at this step with the polynucleotides oO Sfrom the sexual PCR process and optionally subjected to one or more additional mc sexual PCR cycles.

SWhere insertion of mutations into the template polynucleotide is desired, N 10 single-stranded or double-stranded polynucleotides having a region of identity to the template polynucleotide and a region of heterology to the template polynucleotide may be added in a 20 fold excess by weight as compared to the total nucleic acid, more preferably the single-stranded polynucleotides may be added in a 10 fold excess by weight as compared to the total nucleic acid.

Where a mixture of different but related template polynucleotides is desired, populations of polynucleotides from each of the templates may be combined at a ratio of less than about 1:100, more preferably the ratio is less than about 1:40. For example, a backcross of the wild-type polynucleotide with a population of mutated polynucleotide may be desired to eliminate neutral mutations mutations yielding an insubstantial alteration in the phenotypic property being selected for). In such an example, the ratio of randomly provided wild-type polynucleotides which may be added to the randomly provided sexual PCR cycle hybrid polynucleotides is approximately 1:1 to about 100:1, and more preferably from 1:1 to 40:1.

The mixed population of random polynucleotides are denatured to form single-stranded polynucleotides and then re-annealed. Only those single-stranded polynucleotides having regions of homology with other single-stranded polynucleotides will re-anneal.

The random polynucleotides may be denatured by heating. One skilled in the art could determine the conditions necessary to completely denature the doublestranded nucleic acid. Preferably the temperature is from 80 OC to 100 more preferably the temperature is from 90 °C to 96 other methods which may be used to denature the polynucleotides include pressure (36) and pH.

The polynucleotides may be re-annealed by cooling. Preferably the 00 temperature is from 20 OC to 75 more preferably the temperature is from 40 °C to 65 If a high frequency of crossovers is needed based on an average of only 4 consecutive bases of homology, recombination can be forced by using a low Sannealing temperature, although the process becomes more difficult. The degree of renaturation which occurs will depend on the degree of homology between the population of single-stranded polynucleotides.

Renaturation can be accelerated by the addition of polyethylene glycol or salt. The salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mm. The salt may be KCl or NaCI. The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to The annealed polynucleotides are next incubated in the presence of a nucleic acid polymerase and dNTP's dATP, dCTP, DGTP and dTTP). The nucleic acid polymerase may be the Klenow fragment, the Taq polymerase or any other DNA polymerase known in the art.

The approach to be used for the assembly depends on the minimum degree of homology that should still yield crossovers. If the areas of identity are large, Taq polymerase can be used with an annealing temperature of between 45-65 If the areas of identity are small, Klenow polymerase can be used with an annealing temperature of between 20-30 OC. One skilled in the art could vary the temperature of annealing to increase the number of cross-overs achieved.

The polymerase may be added to the random polynucleotides prior to annealing, simultaneously with annealing or after annealing.

66 The cycle of denaturation, renaturation and incubation in the presence of polymerase is referred to herein as shuffling or reassembly of the nucleic acid. This cycle is repeated for a desired number of times. Preferably the cycle is repeated from 2 to 50 times, more preferably the sequence is repeated from 10 to 40 times.

00 Mc The resulting nucleic acid is a larger double-stranded polynucleotide of from about 50 bp to about 100 kb, preferably the larger polynucleotide is from 500 bp to kb.

N This larger polynucleotides may contain a number of copies of a polynucleotide having the same size as the template polynucleotide in tandem. This concatemeric polynucleotide is then denatured into single copies of the template polynucleotide. The result will be a population of polynucleotides of approximately the same size as the template polynucleotide. The population will be a mixed population where single or double-stranded polynucleotides having an area of identity and an area of heterology have been added to the template polynucleotide prior to shuffling.

These polynucleotides are then cloned into the appropriate vector and the ligation mixture used to transform bacteria.

It is contemplated that the single polynucleotides may be obtained from the larger concatemeric polynucleotide by amplification of the single polynucleotide prior to cloning by a variety of methods including PCR (USPN 4,683,195 and USPN 4,683,202), rather than by digestion of the concatemer.

The vector used for cloning is not critical provided that it will accept a polynucleotide of the desired size. If expression of the particular polynucleotide is desired, the cloning vehicle should further comprise transcription and translation signals next to the site of insertion of the polynucleotide to allow expression of the polynucleotide in the host cell. Preferred vectors include the pUC series and the pBR series of plasmids.

The resulting bacterial population will include a number of recombinant polynucleotides having random mutations. This mixed population may be tested to identify the desired recombinant polynucleotides. The method of selection will depend on the polynucleotide desired.

oO c For example, if a polynucleotide which encodes a protein with increased binding efficiency to a ligand is desired, the proteins expressed by each of the Sportions of the polynucleotides in the population or library may be tested for their ability to bind to the ligand by methods known in the art panning, affinity chromatography). If a polynucleotide which encodes for a protein with increased drug resistance is desired, the proteins expressed by each of the polynucleotides in the population or library may be tested for their ability to confer drug resistance to the host organism. One skilled in the art, given knowledge of the desired protein, could readily test the population to identify polynucleotides which confer the desired properties onto the protein.

It is contemplated that one skilled in the art could use a phage display system in which fragments of the protein are expressed as fusion proteins on the phage surface (Pharmacia, Milwaukee WI). The recombinant DNA molecules are cloned into the phage DNA at a site which results in the transcription of a fusion protein a portion of which is'encoded by the recombinant DNA molecule. The phage containing the recombinant nucleic acid molecule undergoes replication and transcription in the cell. The leader sequence of the fusion protein directs the transport of the fusion protein to the tip of the phage particle. Thus the fusion protein which is partially encoded by the recombinant DNA molecule is displayed on the phage particle for detection and selection by the methods described above.

It is further contemplated that a number of cycles of nucleic acid shuffling may be conducted with polynucleotides from a sub-population of the first population, which sub-population contains DNA encoding the desired recombinant protein. In this manner, proteins with even higher binding affinities or enzymatic activity could be achieved.

It is also contemplated that a number of cycles of nucleic acid shuffling may be conducted with a mixture of wild-type polynucleotides and a sub-population of oO nucleic acid from the first or subsequent rounds of nucleic acid shuffling in order to Cc remove any silent mutations from the sub-population.

c-I SAny source of nucleic acid, in purified form can be utilized as the starting CI 10 nucleic acid. Thus the process may employ DNA or RNA including messenger RNA, which DNA or RNA may be single or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. The nucleic acid sequence may be of various lengths depending on the size of the nucleic acid sequence to be mutated. Preferably the specific nucleic acid sequence is from 50 to 50000 base pairs. It is contemplated that entire vectors containing the nucleic acid encoding the protein of interest may be used in the methods of this invention.

The nucleic acid may be obtained from any source, for example, from plasmids such a pBR322, from cloned DNA or RNA or from natural DNA or RNA from any source including bacteria, yeast, viruses and higher organisms such as plants or animals. DNA or RNA may be extracted from blood or tissue material.

The template polynucleotide may be obtained by amplification using the polynucleotide chain reaction (PCR, see USPN 4,683,202 and USPN 4,683,195).

Alternatively, the polynucleotide may be present in a vector present in a cell and sufficient nucleic acid may be obtained by culturing the cell and extracting the nucleic acid from the cell by methods known in the art.

Any specific nucleic acid sequence can be used to produce the population of hybrids by the present process. It is only necessary that a small population of hybrid sequences of the specific nucleic acid sequence exist or be created prior to the present process.

The initial small population of the specific nucleic acid sequences having mutations may be created by a number of different methods. Mutations may be Screated by error-prone PCR. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. Alternatively, mutations can be introduced into the template 00 polynucleotide by oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the N polynucleotide using restriction enzyme digestion and is replaced with a synthetic Spolynucleotide in which various bases have been altered from the original sequence.

IN 10 The polynucleotide sequence can also be altered by chemical mutagenesis. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. other agents which are analogues of nucleotide precursors include nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally, these agents are added to the PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.

Generally, plasmid polynucleotides so mutagenized are introduced into E. coli and propagated as a pool or library of hybrid plasmids.

Alternatively the small mixed population of specific nucleic acids may be found in nature in that they may consist of different alleles of the same gene or the same gene from different related species cognate genes). Alternatively, they may be related DNA sequences found within one species, for example, the immunoglobulin genes.

Once the mixed population of the specific nucleic acid sequences is generated, the polynucleotides can be used directly or inserted into an appropriate cloning vector, using techniques well-known in,the art.' The choice of vector depends on the size of the polynucleotide sequence and the host cell to be employed in the methods of this invention. The templates of this invention may be plasmids, phages, cosmids, phagemids, viruses retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof coat protein, spike glycoprotein, capsid protein). For example, cosmids and phagemids are preferred where the specific nucleic acid sequence to be mutated is larger because these vectors are able to stably propagate large polynucleotides.

CN If the mixed population of the specific nucleic acid sequence is cloned into a O vector it can be clonally amplified by inserting each vector into a host cell and CN 10 allowing the host cell to amplify the vector. This is referred to as clonal amplification because while the absolute number of nucleic acid sequences increases, the number of hybrids does not increase. Utility can be readily determined by screening expressed polypeptides.

The DNA shuffling method of this invention can be performed blindly on a pool of unknown sequences. By adding to the reassembly mixture oligonucleotides (with ends that are homologous to the sequences being reassembled) any sequence mixture can be incorporated at any specific position into another sequence mixture.

Thus, it is contemplated that mixtures of synthetic oligonucleotides, PCR polynucleotides or even whole genes can be mixed into another sequence library at defined positions. The insertion of one sequence (mixture) is independent from the insertion of a sequence in another part of the template. Thus, the degree of recombination, the homology required, and the diversity of the library can be independently and simultaneously varied along the length of the reassembled DNA.

This approach of mixing two genes may be useful for the humanization of antibodies from murine hybridomas. The approach of mixing two genes or inserting alternative sequences into genes may be useful for any therapeutically used protein, for example, interleukin I, antibodies, tPA and growth hormone. The approach may also be useful in any nucleic acid for example, promoters or introns or 31 untranslated region or 51 untranslated regions of genes to increase expression or V alter specificity of expression of proteins. The approach may also be used to mutate ribozymes or aptamers.

Shuffling requires the presence of homologous regions separating regions of diversity. Scaffold-like protein structures may be particularly suitable for shuffling.

00 The conserved scaffold determines the overall folding by self-association, while displaying relatively unrestricted loops that mediate the specific binding. Examples of such scaffolds are the immunoglobulin beta-barrel, and the four-helix bundle which are well-known in the art. This shuffling can be used to create scaffold-like 10 proteins with various combinations of mutated sequences for binding.

Saturation mutagenesis In one aspect, this invention provides for the use of proprietary codon primers (containing a degenerate N,N,G/T sequence) to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full oO range of single amino acid substitutions is represented at each amino acid position.

C

c The oligos used are comprised contiguously of a first homologous sequence, a

C

I degenerate N,N,G/T sequence, and preferably but not necessarily a second O homologous sequence. The downstream progeny translational products from the use c 10 of such oligos include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions. In another aspect, at least two degenerate N,N,G/T cassettes are used either in the same oligo or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions. Thus, more than one N,N,G/T sequence can be contained in one oligo to introduce amino acid mutations at more than one site. This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s). In another aspect, oligos serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.

In a particular exemplification, it is possible to simultaneously mutagenize two or more contiguous amino acid positions using an oligo that contains contiguous N,N,G/T triplets, i.e. a degenerate sequence.

In another aspect, the present invention provides for the use of degenerate cassettes having less degeneracy than the N,N,G/T sequence. For example, it may be desirable in some instances to use in an oligo) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet. Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet. Alternatively, it may be desirable in some instances to use in an oligo) a degenerate N,N,N triplet 00 sequence, or an N,N, G/C triplet sequence.

O

C It is appreciated, however, that the use of a degenerate triplet (such as SN,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instant invention is CI 10 advantageous for several reasons. In one aspect, this invention provides a means to systematically and fairly easily generate the substitution of the full range of possible amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide. Thus, for a 100 amino acid polypeptide, the instant invention provides a way to systematically and fairly easily generate 2000 distinct species (i.e.

20 possible amino acids per position X 100 amino acid positions). It is appreciated that there is provided, through the use of an oligo containing a degenerate N,N,G/T or an N,N,G/C triplet sequence, 32 individual sequences that code for 20 possible amino acids. Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using one such oligo, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides. In contrast, the use of a non-degenerate oligo in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel.

This invention also provides for the use ofnondegenerate oligos, which can optionally be used in combination with degenerate primers disclosed. It is appreciated that in some situations, it is advantageous to use nondegenerate oligos to generate specific point mutations in a working polynucleotide. This provides a means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.

Thus, in a preferred embodiment of this invention, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide molecules such that all 20 amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide. The 32-fold degenerate progeny polypeptides generated 00 from each saturation mutagenesis reaction vessel can be subjected to clonal

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eC amplification cloned into a suitable E. coli host using an expression vector) and CI subjected to expression screening. When an individual progeny polypeptide is O identified by screening to display a favorable change in property (when compared to CN 10 the parental polypeptide), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3 x 3 x 3 or 27 total possibilities, including 7 that were previously examined 6 single point mutations 2 at each of three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used together with shuffling, chimerization, recombination and other mutagenizing processes, along with screening. This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner. In one exemplification, the iterative use of any mutagenizing process(es) is used in combination with screening.

Thus, in a non-limiting exemplification, this invention provides for the use of saturation mutagenesis in combination with additional mutagenization processes, such as process where two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment.

In addition to performing mutagenesis along the entire sequence of a gene, the instant invention provides that mutagenesis can be use to replace each of any Snumber of bases in a polynucleotide sequence, wherein the number of bases to be N1 mutagenized is preferably every integer from 15 to 100,000. Thus, instead of Smutagenizing every position along a molecule, one can subject every a discrete C1 10 number of bases (preferably a subset totaling from 15 to 100,000) to mutagenesis.

Preferably, a separate nucleotide is used for mutagenizing each position or group of positions along a polynucleotide sequence. A group of 3 positions to be mutagenized may be a codon. The mutations are preferably introduced using a mutagenic primer, containing a heterologous cassette, also referred to as a mutagenic cassette. Preferred cassettes can have from 1 to 500 bases. Each nucleotide position in such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E can be referred to as a designer oligo). The tables below show exemplary trinucleotide cassettes (there are over 3000 possibilities in addition to N,N,G/T and N,N,N and N,N,A/C).

In a general sense, saturation mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is preferably 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is preferably from 15 to 100,000 bases in length).

Thusly, a group of mutations (ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized. A grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of saturation mutagenesis. Such groupings are exemplified by deletions, additions, groupings of particular codons, and groupings of particular nucleotide cassettes.

Defined sequences to be mutagenized (see Fig. 20) include preferably a whole gene, pathway, cDNA, an entire open reading frame (ORF), and entire promoter, enhancer, repressor/transactivator, origin of replication, intron, operator, or any polynucleotide functional group. Generally, a preferred "defined sequences" for this purpose may be any polynucleotide that a 15 base-polynucleotide sequence, oO Sand polynucleotide sequences of lengths between 15 bases and 15,000 bases (this

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0C invention specifically names every integer in between). Considerations in choosing C groupings of codons include types of amino acids encoded by a degenerate 0 mutagenic cassette.

CN In a particularly preferred exemplification a grouping of mutations that can be introduced into a mutagenic cassette (see Tables 1-85), this invention specifically provides for degenerate codon substitutions (using degenerate oligos) that code for 2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids at each position, and a library of polypeptides encoded thereby.

TABLE Site I Site 2 Site 3 #ofa".'s NPL: POL NEC I. N,N.CIT N N GiT 20 IS: 9: 2 5: 1 2. NN.G/C N N G/C 20 15: 9: 2: 5: 1 3. N.N.G/A N N 6/A 14 IS: 6: 2: 6: 3 4. N.NA/C N N A/C 18 14: 9: 2: 5: 2 NN.A/T N N AT I 1s 14: 9: 2: 5: 2 6. NN.CT N N CfT IS 14: 12: 2 4: 0 7. NN,N N N ;N 20 29: Is: 4: 10: 3 8. N.N.C N N U 13 8: 3: 1: 3 9. NNA N N A 12 7: 3: 1: 3 2 NN,C N N C IS 7: 6: 1: 2 0 II. N,NT N N T IS 7: 6: 1: 2: 0 12. N.N.C/G/T N N C/CIT 20 22: IS: 3: 7: 1 13. NN.A/G/T N N A/r/T 20 22: 12: 3: 80 3 14. NN,A/C/I N N A/CT 1S 21: IS: 3: 7: 2 N,NA/C/G N N A/C/G 20 22: 12: 3: 8: 3 16. N.A.A N A A 3 0: I: I: I: 17. NAC N A C 4 0: 2: I: I 0 18. N.A.C N A G 3 0: I: I: I: 1 19. N.A.T N A T 4 0: 2: I: I 0 NC.A N C A 4 2: 2: 0: 0 0 21. NC.C N C C 4 2: 2: 0: 0: 0 22. NC,G N C C 4 2: 2: 0: 0: 0 23. N,C.T N C T 4 2: 2: 0: 0: 0 24. N.G.A N G A 2 I: 0: 0: 2: 1 N,G.C N G C 4 I: 2: 0: I 0 26. N,G,G N G G 3 2: 0: 0 2: 0 27. N.C.T N G T 4 I: 2: 0: I 0 28. N.T.A N T A 3 4: 0: 0: 0: 0 29. N.TC N T C 4 4: 0: 0: 0: 0 N,T.G N T C 3 4: 0: 0: 0: 0 31. NT.T N T T 4 4: 0: 0: 0: 0 32. NA/CA N A/C A 7 2: 3: 1: I 33. N.A/.A N A/C A 5 I: I: 1: 3: 2 34. N.A/TA N A/T A 6 4: 1: I: I N.C/G.A N C A 6 3: 2: 0: 2: I 36. NCT.A N A 7 ur A 7 6: 2: 0: 0: 0 37. N,T/CA N T/C A 5 5: 0: 0: 2: 38. NC/C.A N C/GIT A 9 7: 2: 0: 2: 39. NA//TA N A/C/T A 8 5: 1: I: 3: 3 NA/C/T.A N AlC/T A 10 6: 3: 1: I 41. N.A/C/CA N A/C/C A 9 3: 3: 1: 3: 2 42. A,N.N 'A N N 7 4: 8: 0: 4: 0 43. CW.N C N N 5 8: 2: 0: 6: 0 44. C.NN G N N4 5 12: 0: 4: 0: 0 T.N.N T N N1 6 5: 8: 01 0: 3 46. A/C.N.N A/C N N ii 12: 10: 0: 10: 0 47. A/C.N.N A/C N N 12 16: 8: 4: 4: 0 48. AT.N.N AIT N N 12 9: 16: 0: 4 3 49. C/G,N.N C/C N N 10 20: 2: 4: 6: 0 CT.NN CIT N .N 10 13: 10: 0: 6: 3 51. G.N.N C/T N N 11 17: 8: 4: 0 3 52. NA,N N A N 7 0: 6: 4: 4: 2 53. N.C.N N C N 4 8: 8: 0: 0: 0 54. N.G.N N G N 5 5: 4: 0: 6: N.T.N N T N 5 16: 0: 0: 0: 0 56. N,A/C.N N A/C N 11 8: 14: 4: 4: 2 57. NA/,N N A/C N 12 5: 10: 4: 10: 3 58. N.A/TN N A/T N 12 16: 6: 4: 4: .59. N.C/C,N N C/C N 8 13: 12: 0: 6: N.C/T.N N C/T N 9 24: 8: 0: 0: 0 61. N.CIT.N N CIT 10 21: 4: 0: 6: 1 62. N.A/C/CN N A/C/G N IS 13: 18: 4: 10: 3 63. NAIC/T.N N A/CI T N 16 24: 14: 4: 4: 2 64. N.A/C/T.N N A/CT Nr 17 21: 10: 4: 10: 3 N.C/C/T.N N C/CTr N 13 29: 12: 0: 6: 66. CC.N c c N I 4: 0: 0: 0 0 67. G.G.N G N I 1 4: 0: 0: 0: 0 68. GC,N C c N I 4: 0: 0: 0 0 69. CT.N C T N I 4: 0: 0: 0: 0 CG.N c C N I 0: 0: 0: 4: 0 71. C.T.N 72. TC.N 73. A.C.N

T

A

T

C

C

'Nt

N

4: 0: 0: 0: 0 0: 0: 0 0: 0: 0 TABLE Site I Site 2 Site 3 #of a.'s NPL Pnlt: N F Q 74. G.A,N GA N4 2 0: 0: 4: 0: 0 A.T.N A T-f N 2 4: 0: 0: 0: 0 76. C.A.N C A N 2 0: 2: 0: 2: -0 77. TT.N T T N 2 4: 0: 0: 0: 0 78. AA.N A A N i 2 0: 2: 0: 0.

7.TANTA N 1 0: 2: 0: 0: 2l T.G.N T G N 2 I 2 0: 0: 1 SI. A,GN A G N 2 0: 2: 2: 0 82. G/C,G,N 0-j -N 24: 0: 0: 4: 0 83. GIC.C.N (i/C C f N 2 8: 0: 0: 0: 0 84 AN I /C A N 4 0: 2: 4: 2: 0 G/CT.N I 0/C T N 2 8 0: 0: 0 TABLE 1. Mutagenic Cassette: N, N, G/T CODON iRepreseunted AMINO ACID (Frequency) CATEGORY (Frequency) GOT YES GLYCLNE 2 NONPOLAR 1s GGC NO

(NPL)

GGA NO 000 YES OCT YES ALANINE 2 0CC NO GCA NO OCG YES GTT YES VALP.JE2 GTC NO GTA NO OTO YES TANO LEUCLNE 3 TTG YES CiT YES CTC NO CTA NO CTO YES ATT YES ISOLEUCINE I ATC NO ATA NO ATG YES METHIONINE 11 m YES PHENYLALANINE I TTC NO TOG YES TRYPTOPHAN CCT YES jPROLINE2 CCC NO J CCA NO J CCG YES TCT YES SERINE 3 POLAR 9 TCC NO

NONIONIZAB

TCA NO

LE

TCO YES

(POL)

AGT YES AOC NO TOT YES CYSTEINEI TOC NO AAT YES ASPARAGINEI AAC NO CAA NO GLUTAMINEI CAG YES TAT YES Th'OSINEI TAC- NO ACT YES THREON-INE 2 ACC NO ACA NO ACO YES GAT YES ASPARTIC ACID I IONIZABLE. 2 GAC NO

ACIDIC

OAR NO GOL LTA M C AC ID I NEGATIVE GAO YES CHARGE

(NEG)

AAR NO LN SINE I IONIZABLE: AAG YE S

BASIC

COT YES ARGININE 3 POSITIVE COC NO CHARGE CG-A NO COO YES AGA NO AGG YES CAT YES HISTIDINEI CAC NO I IAR NO ISTOPCODON I STOPSIGNAL I TAO YES J(STn) TOA NO TOTAL 64 3220 Amino Acids Are NI'L: POL: NEC: POS: Represented STP IS1: 9: 2: TABLE 2. Mutagenic Cassette: N, N, GIC CODON Represened AMINO ACID (Frequency) CATEGORY (Frequency) GGT NO OLYCINE 2 NONPOLAR I GGC YES

(NPL)

OGA NO COG YES OCT NO ALANINE 2 GCC YES GCA NO GCG YES iT NO VALINE 2 GTC YES OTA NO GTG YES TTA NO LEUCINE 3 TTG YES CT NO CTC YES CTA NO CTG YES ATT NO ISOLEUCINE ATC YES ATA NO ATG YES METHIONINE iTT NO PHENYLALANINE

I

TC YES TGG YES TRYPTOPHAN CCT NO PROLINE 2 CCC YES CCA NO CCC YES TCT NO SERINE 3 POLAR 9 TCC YES

NONIONIZABLE

TCA NO

(POL)

TCY

AGT NO AGC YES TGT NO CYSTEINE

I

TGC YES AAT NO ASPARAGINE

AAC

CAR NO GLUTAMINE

I

CAGYES

TAT NO TYROSINE TAC YES ACT NO THREONINE ACC YES ACA N ACG YES GAT NO ASPARTIC ACID I IONIZABLE- ACIDIC 2 GAC YES NEGATIVE CHARGE GAR NO OLUTAMIC ACID

(NEG)

GAGYE

AAA NO LYSINE I IONIZABLE: BASIC AAG YES POSmnVE CHARGE CGT NO ARG1NINE 3 (POS)

CCYES

COA NO CGG YES AGA NO AGO YES CAT NO HISTIDINE I CAC YES 'TAR NO STOP CODON I STOP SIGNAL TAO YES

(SW)

TGA NO TOTAL TOTAL 20 Amino Acids Are Represented NFL: POL: NEG: POS: STP- 64 32 1 IS: 9: 2: 5: 1 TABLE 3. Mutagenic Cassette: N, N, G/A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGT NO GLYCINE 2 NONPOLAR IS GGC NO (NPL) OGA YES GGG YES OCT NO ALANINE 2 0CC NO OCA YES GCO YES GTT NO VALINE 2 GTC NO GTA YES OTO YES ITA YES LEUCINE 4 TTG YES cr'r NO CTC NO CTA YES

YES

AlT NO ISOLEUCINE I ATC NO ATA YES ATO YES METHIONINE TTT NO PHENYLALANINE 0 TTC NO TGG YES TRYPTOPHAN CCT NO PROLINE 2 CCC NO CCA YES CCG YES TCT NO SERINE 2 POLAR 6 TCC NO NONIONIZABLE TCA YES (POL) TCG YES

AOT

AGC NO TOT NO CYSTEINE 0 TGC NO AAT NO ASPARAGINE 0 AAC CAA YES GLUTAMINE 2

CAGYES

TAT NO TYROSINE 0 TAC NO ACT NO THREONINE 2 ACC NO ACA YES ACG YES GAT NO ASPARTIC ACID 0 IONIZABLE- ACIDIC 2 GAC NO NEGATIVE CHARGE OAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 6 AAO YES POSITIVECHARGE CGT NO ARGININE 4 (POS) CGC NO CGA YES CGG YES AGA YES AGO YES CAT NO HISTIDINE 0 CAC NO TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES (STP) TGA YES TOTAL TOTAL 14 Amino Acids Are Represented NPL; POL:NEG: POS: STP- 64 32 15: 6: 2: 6: 3 TABLE 4. Mutagenic Cassette: N, N, A/C CODON fRepresented j AMINO ACID (Frequency) CATEGORY (Frequency) -GGT NO -LCN NONPOLAR 14 YGC ES

(NPL)

GGG NO OCT NO ALANINE 2

GCCYE

GCA E GTT NO VALINE 2 GTC YES GTA YES GIG NO TTA YES LEUCINE 3 T NO CTT NO CTC YES CTA YES CTG NO ATT NO ISOLEUCINE 2 ATC YES ATA YES NO METHIONINE 0 TT I NO PHENYLALANINE I T YES TG I NO TRYPTOPHAN 0 CCT NO PROUNE 2 CCC j YES CCA 1 YES CCG NO TCT NO SERINE 3 POLAR 9 TCC YES

NONIONIZABLE

TCA YES

(POL)

TCO NO ACT NO AGC YES TGT NO0 CYSTEINEI TGC YES AAT NO ASPARAGINE I AAC YES CAA YES GLUTAMINEI CAG NO TAT NO TYROSINE I TAC YES ACT NO ITHREONINE 2 ACC YES AC-A YES ACG NO GAT NO ASPARTIC ACID I IONIZABLE: ACIDIC 2 GAC YES NEGATIVE CHARGE OAA YES j LUTAMIC ACID I (NEC) GAG NO 1 AAA YES LYSINE I IONIZABLE: BASIC 3 AAO NO POSITIVE CHARGE CGT NO ARGININE

(O)

CGC YES CGA YES CGG NO- AGA YES AGG No CAT NO HISTIDINEI CAC I YES 'rAA j YES JSTOP CODON 2 STOP SIGNAL 2 TAG j NO J(SIP) TGA I YES I OTAL ITOTAL IIS Amino Acids Are Represented NPL: FOL: NEG: POS: STP 64 32 7 14: 9: 2: 5: 2 TABLE 5. Mutagenic Cassette: N, N, A/T CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GOT YES (JLYCINE 2 NONPOLAR 14 (36C NO (NPL) GGA YES 600 NO GCT YES ALANINE 2 GCC NO GCA YES GCG NO GTr YES VALINE 2 OTC NO GTA YES OTO NO TI7A YES LEUCINE 3 iTO NO CTT YES CTC NO CTA YES CTG NO ATT YES ISOLEUCINE 2 ATC NO ATA YES ATO NO METHIONINE 0 iTT YES PHENYLALANINE I TTC NO TGG NO TRYPTOPHAN 0 CCT YE PROLINE. 2 CCC

EENO

CCA ES CCG No TCT YES SHRINE 3 POLAR 9 TCC NO NONIONIZABLE TCA YES (POL) TCO NO AGT YES AGC NO TGT YES CYSTEINEI TGC NO AAT YES ASPARAGINEI AAC NO CAA YES GLUTAMINE I CAG NO TAT YES TYROSINE. I TAC NO ACT YES TIIREONINE2 ACC NO ACA YES J ACG NO OAT YES IASPARTIC ACID I IONIZABLE: ACIDIC 2 GAC NO I NEGA7IVE CHARGE GAA YES" GLUTAMIC ACID I (NEG) GAG NO AAA YES ILYSINE I IONIZABLE: BASIC AAG NO 1 osmvE CHARGE COT YES ARGININE 3 (PO) COC NO COA YES COG NO AGA YES AGO NO CAT Y ES HISTIDINEI CAC NO TAA YES jSTOP CODON2STPIGA2 TAO NO

S)

TOA YES 1 TOTAL TOTAL 18 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 327 1: 9 2: 5 2 TABLE 6. Mutagenic Cassette: N, N, C/T CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES GjLYCINE 2 NONPOLAR 14 GGC YES (NPL) GGA NO GGG NO OCT YES ALANINE 2 0CC YES GCA NO GCG NO GTT YES VALINE 2 GTC YES GTA NO GTG NO TTA NO LEUCINE 2 TTG NO CIT YES CTC YES CTA NO CTG NO ATT YES ISOLEUCINE 2 ATC YES ATA NO ATO NO METHIONINE 0 TTYES j PHENYLALANINE 2 T'TC YES TOO NO TRYPTOPHAN 0 CCT YES PROLINE 2 CCC YES CCA NO CCO NO TCT YES SERINE 4 POLAR 12 TCC NONIONIZABLE TCA NO

(POL)

TCO NO AGT YES AGC YES TGT YES CYSTINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YE 1 CAA NO GLUTAMINE 0 CAG NO TAT YES J TYROSINE 2 TAC YES ACT YES T THREONINE 2 ACC YES ACA NO ACG NO OAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 2 GAC YES 1 NEGATIVE CHARGE GAA NO GLUTAMIC ACID 0 (NEG) GAG I NO AAA NO LYSINE 0 IONIZABLE: BASIC 4 AAC NO 1 POSTIVE CHARGE CGT YES ARGININE 2 (POS) CGC YES CGA NO CGO NO AGA NO

AGO

CAT YES HISTIDINE 2 CAC L YES TAA NO STOPCODON 0 STOP SIGNAL 0 TAG NO

(STP)

TGA NO TOTAL TOTAL 15 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 32 14: 12: 2: 4: 0 TABLE 7. Mutagenic Cassette: N, N, N CODON !Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES OLYCINE 4 NONPOLAR 29 GOC YES (NPL) G00 YES OCT YES ALANINE 4 GCC YES GCA YES GCG YES GTr YES VALINE 4 GTC YES GTA YES GTO YES TTA YES LEUCINE 6 1-ro YES cTr YES CTC YES CTA YES CTG YES ATT E ISOLEUCIINE 3

ATCYE

ATAYE

ATG YES jMETHIONINEI 1T YES jPHENYLALANINE 2 iT-r YES TOG YES TRYPTOPHANI CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES SERINE 6 POLAR is TCC YES NONIONIZABLE TCA YES (POL) TCO E AGT YES AGC YES TOT YES CYSTEINE 2 TGC YES AAT YES ASPARAOINE 2 AAC YES CAA YES OLUTAMINE 2

CAGYE

TAT YES TYROSINE2 TAC YES ACT YES THRE)NINE 4 ACC YES ACA YES ACG YES GAT YES I ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE OAA YES G LUTANIIC ACID 2 (NEG) GAG YES 1 AAA YES I LYSINE 2 IONIZABLE: BASIC AAG YES POSITIVE CHARGE COT YES AROININE 6 (POS) COC YES CGA YES COO YES AGA YES AGO YES CAT YE HISTIDINE 2 CAC YES I TAA YES I STOP CODON 3STOP SIGNAL 3 TAG YES J(STh) TGA YES TOTAL TOTAL 120 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 64 J29: 18: 4: 10: 3 TABLE 8. Mutagenic Cassette: N, N, G CODON Represente AMINO ACID (Frequency) CATEGORY (Frequency) GOT NO _jGLYCINE

INNOA

GGC NO(NPL) 666 YES OCT NO ALANINEI OCC NO GCA NO GCG YES G NO VALINEI GTC NO GTA NO GTG YES TrA NO LEUCINE 2 r YES CTC N CTA NO CTG YES ATT NO ISOLEUCINE 0 ATC NO ATA NO ATO YES LMETHIONINEI Tn NO [PHENYLALANINE 0 lTT NO TOO YES TRYPTOPHANI CCT No MROLINE I CCC No CCA ENO CCG YES TCT NO SHRINE I POLAR 3 TCC NO0

NONIONIZABLE

TCA NO

(POL)

TCG YES AGT N TGT NO CYSTEINE 0 TOC NO AAT NO ASPARAOINE 0 AAC NO CAA NO GLUTAMINE CAG YES TAT NO TYROSINE 0 TAC NO ACT NO THREONINE I ACC NO ACA N ACO YES GAT NO ASPARTIC ACID 0 IONIZABLE. ACIDIC GAC NOEE NEGATIVE CHARGE OAA NO GLUTANIIC ACID I (NEG) GAG YES AAA LYIEI IONIZABLE: BASIC 3 AAG YES POSITIVE CHARGE COT NO ARGININE COC NO CGA NO CGO YES AGA NO AGO YES CAT ND HISTIDINE 0I CAC NO 'FAA NO STOP CODON I STOP SIGNALI TAG YES J(ST?) TGA NO I TOTAL TOTAL 13 Amino Acids Are Represented NPL: POLUNEG: pos: STP: 64 16 1 8: 3: 1: 3: 1 TABLE 9.__Mutagenic Cassette: N, N, A CODON Represented AMINO ACID (Frequency) CATEGO0RY (Frequency) GGT NO- GLYCINE I NONPOLAR 7 GGC NO (NPL) OGA YES GGG NO GCT NO ALANINEI 0CC NO OCA YES GCG NO GTT NO VALINEI GTC NO OTA YES OTG NO TI'A YES LEUCINE 2 iTO NO CiT NO CTC NO CTA YES CT0 NO ATr NO ISOLEUCINE I ATC NO ATA YES ATG NO METHIONINE 0 liT NO PHENYLALANINE 0 TC NO TGG NO TRYPTOPI4AN 0 CCT NO PROLINE I CCC NO CCA YES CCG NO TCT NO SERINE I POLAR 3 TCC NO NONIONIZABLE TCA YES (POW) TCG NO AGT NO AOC NO TOT NO CYSTETNE 0 TGC NO AAT NO ASPARAGINE 0 AAC NO CAA YES GLUTAMINE I CAG NO TAT NO TYROSINE 0 TAC NO ACT NO THREONINE I ACC NO ACA YES ACO NO GAT NO ASPARTIC ACID 0 IONIZABLE-~ ACIDICI GAC NO NEGATIVE CHARGE GAA YES OLUTAMIC ACID I (NEG) GAG NO AAA Y ES LYSINE I IONIZABLE: BASIC 3 AAO NO POSITIVE CHARGE COT NO ARGININE 2 (POS) CGC NO COA YES COO NO AGA YES AGO NO CAT N HISTIDINE0 CAC NO TAA YES JSTOP CODON 2 STOP SIGNAL 2 TAG NO J(SiT) TOA I YES TOTAL TOTAL 12 Amino Acids Are Represented NPL: POL. NEG: POS: STP 6426 1 7: 3: 1 3: 2 TABLE 10. Mutagenic Cassette: N, N, CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGT NO GLYCINE I NONPOLAR 7 GOC YES

(NPL)

GGA NO 066 NO OCT NO ALANINE I

OCCYE

GCA N OCO NO GITrN VAUINE cJTCYS GTA N TTA NO LEUCINE TO NO CiT NO

CTCYE

CTA NO CTG NO ATT NO ISOLEUCINE ATC YES ATA NO ATG NO METIIIONINE 0 iTr NO PIIENYLALANINE I TTC YES TGG NO TRYPTOPHAN 0) CCT NO PROLINEI CCC YES CCA NO CCA NO CC NO TCT NO SERINE 2 POLAR 6 TCC YES NONIONIZABLE TCA NO

(POLW

TCG NO AGT NO AGC YES TOT NO CYSTEINEI TCIC YES ART NO ASPARAGINEI CAR NO J GLUTAMINE 0 CAO

NO]

TAT NO J YRSN TAC MSS d A CT NO JTHREONINE I ACC YES___ ACA NO AC NO GAT NO I ASPARTIC ACID I IONIZABLE: ACIDICI GAC YES NEGATIVE CHARGE OAR NO 1GLUTAMIC ACID 0 (NEO) GA 0 AAA NO ILYSINE 0 IONIZABLE: BASIC 2- ARO NO 1 POSMVE CHARGE COT NO JAROININE I (POS) CGC YES CGA NO COO -NO AGA NO AOO O CAT NO jHISTIDINE I CAC YES 1 TAA NO ISTOP CODON 0 STOP SIGNAL 0 TAG NO J (STP) TOA L NO TOTA TOTAL IS1 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 I 16 1 6: 1: 2: 0 TABLE 11. Mutagenic Cassette: N, N, T CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GOT YES GLYCINE INONPOLAR 7 GOC NO

(NPL)

GGA NO 000 NO OCT YES ALANINEI 0CC NO OCA NO GCG NO GTT YES VALINEI OTC NO OTA NO OTG NO TTA NO LEUCINEI iTO NO CiT YES CTC NO CTA NO CTO NO A77 YES ISOLEUCINEI ATC No ATA NO ATO NO -TMETHIONINE 0 iM YES PHENY LALANINE I TTC NO TOO NO TRYPTOPHAN 0 CCT YES PROLINE

I

CCC j NO CCA NO CCO NO TCT YES ISERINE 2 POLAR TCC NONONIONIZABLE TCA NO(POL) TCO N TOT YES CYSTEINEI TOC NO AAT YES ASPARAGINEI AAC NO CAA NO ]GLUTAMINE 0 CAG NO TAT I YES Th'ROSINE TAC J NO ACT I YES THREONINE

I

ACC I NO ACA NO ACO N GAT j YES JAATCAIDI IONIZABLE: ACIDICI GAC f O 1NEGATIVE

CHARGE

GAA 0 O GLUTAMIC ACID 0 (NEG) GAG N AAA NO JLYSINE 0 IONIZABLE: BASIC 2 AAO NO POSITIVE CHARGE COT YES AROININE I (POS) COC NO COA NO COO NO AGA NO AGO NO CAT YES HISTIDINEI CAC NO fA 4 NO STOP CODO.N 0 STOP SIGNAL 0 TAG 4 NO

(STP)

TOA I NO TOTAL FOTAL 15 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 J 16 1 7: 6: I 2: 0 TABLE 12. Mutagenic Cassette: N, N, CIG/T CODON Represente M AMINO ACID (Frequency) CATEGORY (Frequency) GOT YS GLYCINE 3 NONPOLAR 22 GOC YES (NPL) GGA NO GO0YO OCT YES ALANINE 3 GCC YES GCA NO GCG YES Grr YES VALINE 3 GTC YES GTA NO GTG YES ilTA NO LEUCINE 4 rro YES CIT YES CTC YES CTA NO CTG YES All' YES ISOLEUCINE 2 ATC YES ATA NO ATO YES METHIONINE I ill' YES PIIENYLALANINE 2 1TC YES TOG YES TRYPTOPHAN I CCT YES PROLINE 3 CCC YES CCA NO CCO YES TCT YES SEINE 5 POLAR TCC YES NONIONIZABLE TCA NO (POL) TCG YES AGT YES AGC YES TOT YES CYSTEINE 2 TOC YES AAT YES ASPARAG[NE 2

AACYE

CAA NO GLUTAMINE I CAG YES TAT YES TYOIE2 TAC YS ACT YES THREONINE 3 ACC YES ACA NO ACG YES GAT YES jASPARTIC ACID 2 IONIZABLE. ACIDIC 3 GAC YES__ NEGATIVE CHARGE OAA NO I LUTAMIC ACID I (NEG) GAG YES AAA NO__I LYSINE I IONIZABLE. BASIC 7 AAO YES POSITVE CHARGE COT YES AROININE 4 (POS) CGC YES CGA NO COO YES AGA NO AGO E CAT YS HISTIDINE 2 CAC YES TANO STOP CDNISTOP SIGNAL TAG YES I CDNI(STP) TGA j NO TOTAL jTOTAL 20 Amino Acids Are Represented NPL: POL: NEG: FOS: STP- 64 48 I22: 15: 3: 7: 1 TABLE 13. Mutagenic Cassette: N, N, A/G/T CODON Representd AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES GLYCINE 3 NONPOLAR 22 GGC NO (NPL) GGA YES OCT YES ALANINE 3 GCC NO GCA YES GCG YES GTT YES VALINE 3 GTC NO OTA YES GTG YES TTA YES LEUCINE 170 YES err YES CTC NO CTA YES CTJ YES AlT YES ISOLEUCINE 2 ATC NO ATA YES ATC YES METHIONINEI l YES IPHENYLALANINE I lTT NO TGG YES TRYPTOPHAN CCT YES PROLINE 3 CCC NO CCA YES CCG YES TCT YES I SERINE 4 POLAR 12 TCC NO NONIONIZABLE TCA YES (POL) TCG YES AGT YES AGC NO TGT YES CYSTEINE I TGC NO AAT YES ASPARAGINE AAC NO CAA YES OLUTAMINE 2 CAG YES I TAT YES TYROSINE I TAC NO ACT YES THREONINE ACC N ACA YES

ACYES

OAT YES ASPARTIC ACID I IONIZABLE: ACIDIC 3 NO NEGATIVECHARGE OAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES LYSINE 2 IONIZABLE BASIC 8 AAG YES POSITIVE CHARGE COT YES ARGININE 5 (POS) CGC NO CGA YES CGG YES AGA YES AGG YES CAT YES HISTIDINE I CAC No rAA YES I STOP CODON 3 STOP SIGNAL 3 TAG YES J (STP) TGA YES TOTAL TOTAL 20 Amino Acids Are Represented NPL: POL: NEG: FOS: STP 64 48 22: 12: 3: 8: 3 TABLE 14. Mutagenic Cassette: N, N, A/C/T CODON I Represented I AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES GLYCINE

YES

GOA_ YES

NONPOLAK

(NPL)

GGG NO OCT YES ALANINE 3 GCC YES OCA YES GCO NO GIT YES VAUNE 3 OTC YES GTA YES GTG NO TA YES LEUCINE 4 TOG NO CT7 YES CTC YES CTA YES

CTG

AT YES ISOLEUCINE 3 ATC YES ATA YES ATG NO IETHIONINE 0 Tm YES PHENYLALANINE 2 TC I YES TGO NO TRYPTOPHAN 0 cCT YES PKOLINt CCC YES CCA YES CCG NO

I

TCT YES SEKINE TCC YES TCA YES TCO NO

AGT

IULAK

NONIONIZABLE

(POL)

AGC YES TOT YES CYSTEINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YES CAA YES OLUTAMINE I CAG NO TAT YES TYROSINE 2 TAC YES I Innrvlrrr ACC YES____ ACA y ACG NO OAT I YES ASPARTIC ACID 2 IONIZABLE. ACIDIC 3 GAC YES NEGATIVE CHARGE GA 1 YES GLUTAMIC ACID I (NEG) GAG NO AAA ES LYSINE I IONIZABLE. BASIC 7 AAG NO I POSITVE CHARGE COT YES ARGININE 4 (POS) CGC YES CGA YES CGG NO

YES

AGG NO CAT YES HISTIDINE 2 CAC 1 YES TAA YES j STOP CODON 2 STOP SIGNAL 2 TAG NO J (STP

YES

TOT TOTAL 18 Amino Acids Are Represented NPL: POL: NEG: POS: STP- 64_ 48 I 21: 15: 3: 7: T

I

TABLE 15. Mutagenic Cassette: N, N, AICIG CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGT NO GLYCINE 3 NONPOLAR 22 GOC YES (NPL) GGA YES 000 YES GCT NO ALANINE 3 0CC YES GCA YES GCG YES GTT NO VAUNE 3 GTC YES OTA YES OTO YES TTA YES LEUC[NE TG YES CIT NO CTC YES CTA YES CTO YES ATT NO ISOLEUCINE 2 ATC YES ATA YES ATG YES METHIONINE Trr NO PHENYLALANINE I ITC YES TGG YES TRYPTOPHAN CCT NO PROLINE 3 CCC YES CCA YES CCG YES TCT NO SERINE 4 POLAR 12 TCC YES NONIONIZABLE TCA YES (POL) TCG YES AGT NO AGC YES TGT NO CYSTEINE I TGC YES AAT NO ASPARAGINE I AAC YES CAA YES GLUTAMINE 2 CAG YES TAT NO TYROSINE I TAC YES ACT NO THREONINE 3 ACC YES ACA YES ACG YES GAT NO I ASPARTIC ACID I IONIZABLE. ACIDIC 3 CIAC YES 1 NEGATIVE CHARGE OAA YES GLUTAMIC ACID 2 (NEG)

YES

AAA YES LYSINE 2 IONIZABLE. BASIC 8 AAO YES POSITIVE CHARGE CGT NO j ARGININE 5 (POS) CGC YES CGA YES CGG YES AGA YES AGO YES CAT NO HISTIDINE CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES j (STP) TGA YES TOTAL TOTAL 20 Amino Acids Are Represented NPL: POL: NEG: POS: STP 64 48 1 22: 12: 3: 8: 3 TABLE 16. Mutagenic Cassette: N, A, A CODON TRepresentedl AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPO1AR 0 ALANINE 0 -(NPL) ___VALINE 0 LEUCINE 0- 0- ______METHIONINE 0 _______PHENYLALANINE 0 0 PROLINE 0 SERINE -0 POLARI 0 NONIONIZABLE __________ASPARAGINE 0 (POL) CAA YES GLUTAMINEI TYROSINE 0 THREONINE 0 ACID 0 IONIZABLE ACIDIC I GAA YS GLUTAMIC ACID NEGATIVE CHARGE AAA YES I LYSINE I IONIZABLE: BASICI o POSTVE CHARGE [HISTIDINE 0 (POS) TAA YES jSTOP CODON I STOP SIGNALI I USP TOTAL 4 j3 Amino Acids Are Represented NPL: POL: NEG: POS: STP TABLE 17. Mutagenic Cassette: N, A, C CODON 1Represented] AMINO ACID (Frequency) CATEGORY (Frequency) I GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VAUINE 0 SLEUCINE 0 ISOLEUCINE 0 METHIONINE 0 SPHENYLALANINE 0 ________TRYPTOPHAN0 PROLINE 0 U POLAR 2 TCYSTEINE 0 NONIONIZABLE AAC I YES T ASPARAGINE I- (POL) I GLLITAMINE 0 TAC YES ITYROSINE I (.iAC j YES ASPARTIC ACID I IONIZAB3LE: ACIDIC I

(NEG)

f YIE0 IONIZABLE: BASIC I ARGININE 0 POSITIVE CHARGE CAC YES I HISMIINE I(PS STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 4 I4 Amino Acids Are Represented NPL: POL: NEG: P05: STP _I0: 2: 1: 1: 0 TABLE 18. Mutagenic Cassette: N, A, G CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 __________METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0- SEINE 0 POLARI 0 NONIONIZABLE _________ASPARAGINE 0 (POL) CAG YES 1GLUTAMINE I- _______]TYROSINE 0 1THREONINE 0 ASPARTIC ACID 0 IONIZABILE: ACIDICI GAG YES 1GLUTAMIC ACID NEGATIVE CHARGE I (NEG) AAG YES LYSINE I IONIZABLE. BASICI ARGININE 0 POSITIVE CHARGE HISTIDINE 0 TAG j YES ISTOP CODON I STOP SIGNALI _I (SIP) TTL4 3 Amino AisAeRpsntd NPL: POL: NEG: POS: STP TOTAL~~0 1: 1 cd r ersne 1:1 TABLE 19. Mutagenic Cassette: N, A, T CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency).

GLYCINE 0 NONPOLAR 0 ALINE 0(P ALINE NL 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0

+TRYPTOPIIAN

SERINE 0 P'OLAR 2 AAT j YES ASPARAGINEI OL GLUTAMINE0 TAT YES TYROSINEI 0 GAT YES ASPARTIC ACID I IONIZABLE: ACIDICI GLUTAMIC ACID 0 NEGATIVE CHARGE m i (NEG) LYSINE 0 IONIZABLE. B3ASICI ARGININE 0 POSITVE CHARGE CAT YES IHISTIDINE I (POS) STOP CODON 0 STOP SIGNAL 0 TOTAL 4 I4 Amino Acids Are Represented NPL: POL: NEG: POS: STP _I0: 2: 1: 1: 0 TABLE 20. Mutagenic Cassette: N, C, CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCA YES ALANINE I (NPL) VAUINE 0 0 ISOLEUCINE 0 0 ___________PHENYLALANINE 0 0 CCA f YES PROLINE I TCA YES SERINE I POLAR 2 T ______CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) I _______GLUTAMINE 0 TYROSINE 0 ACA T YES THREONINEI -ASPARTIC ACID IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE LYSINE 0 IONIZABLE BASIC 0 0 POSTVE CHARGE HIST1DINE 0 (POS) TTLSTOP CODON 0STOP SIGNALEG 0 1 2: 2: 0: 0: 0 TABLE 21. Mutagenhc Cassette: N, C, C CODON TRepresented AMINO ACID (Frequency) CATEGORY (Frequency) ______GLYCINE 0 NONPOLAR 2 GCC f YES ALANINE I (NPL) VALINE 0 0 ISOLEUCINE 0 0 ________PHENYLALANINE 0 ITRYPTOPIIAN 0 CCC I YES PROUINEI TCC I YES SEINE I1 POLAR 2 T ______CYSTEINE 0 NONIONIZABLE 0 (POL) TYROSINE 0 ACC T YES THREONINEI ASPARTIC ACID 0 IONIZABLE.- ACIDIC 0

(NEG)

LYSINE 0 IONIZABLE. BASIC 0 ARGININE POSITIVE CHARGE HISTIDINE 0(POS) STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP 4 2: 2: 0: 0: 0 *TABLE 22. Mutagenic Cassette: N, C, C CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE U NONPOLAR 2 GCG YES ALANINE I(NPL) 0 _______LEUCINE 0 I ISOLEUCINE 0 _______METHIONINE 0 PHENYLALANINE 0 ________TRYPTOPHAN

V-

CCG YES P7ROLI NE I TCG YES SERINE I POLAR 2 CYSTEINE 0 NONIONIZABLE 0 (POL) _______GLUTAMINE 0 _______TYROSINE 0 ACG YES THREONINE

I

________IASPARTIC ACID 0 IONIZABLE ACIDIC U TGLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE: BASIC 0 JARGININE 0* POSITIVE CHARGE HISTIDINE 0 (IS STOP CODON 0 STOP SIGNAL 0 (STh) TOTAL j 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP= 2: 0: 0: 0 TABLE 23. Mutagenic Cassette: N, C, T CODON Rept esented AMINO ACID (Frequency) CATEGORY (Frequency) ______GLYCINE 0NONPOLAR 2 GCT YES ALANINE I (NFL) AUNE 0 LEUCINE 0 ________ISOLEUCINE 0- ________METHIONINE 0- ________PHENYLALAN1INE 0 _______TRYPTOPfIAN 0 CCT YES PROLINE

I-

TCT I YES I SERINE I POLAR 2 ICYSTEINE 0 NONIONIZABLE J_______ASPARAGINE 0(POL) ______GLUTAMINE 0 I ______TROSINE 0 ACT j YES ITHREONINE

I

I ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE LYSINE 0 IOIALE AI 0 POSITIVE______ CHARGE STOP CODON 0 STOP SIGNAL 4) I TOTL 4 4 A~noNPL: POL: NEG: POS: STP= TTL4 4AioAcids Are Represented F 2 0 TABLE 24. Mutagenic Cassette: N, G, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) YES GLYCINE I NONPOLAR ALANINE 0 (NPL) 0 LEUCINE 0 ISOLEUCINE 0 METIONINE 0 ________PHENYLALANINE 0 _______TRYPTOPHAN 0 0 0POLAR 0 _______CYSTEIN 0 NONIONIZABLE _______ASPARAGINE 0 (POL) GLUTAMINE 0 0 0 ACID 0I IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE f YIE0 IONIZABLE. BASIC 2 CGA YES ARGININE 2 POSITIVE CHARGE AGA YES

(POS)

0 TGA YES STOP CODON I STOP SIGNAL I- I

(STP)

TOTAL j 4 I2 Amino Acids Are Represented NPL: POL: NEG: POS: STP 0: 0: 2: 1 TABLE 25. Mutagenic Cassette: N, G, C CODON Represene- AMINO ACID (Frequency) CATEGORY (Frequency) GOrC YES GLYCINE I NONPOLARI 0 (NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 ____________PHENYLALANINE 0 TYPTOPHAN 0 0 AGC YES SRGINEI POLAR 2 YES C MSEIN I NONIONIZABLE 0- PL _______TYROSINE 0 CCC YESC 7 A§3CID 0 IONIZABLE. ACIDIC 0 STOP CODON 0 STOP SIGNAL 0 I_ TTL4 4 Amino Acids Are Represented TNPL: POL: NEG: P05: STP I I: 2 0: 1I: 0 TABLE 26. Mutagenic Cassette: N, G, G CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) 00 YES IGLYCINE I NONPOLAR. 2 ALANINE 0(NPL) ______VALINE 0 _______LEUCINE 0 SOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGG YES TRYPTOPHAN

I-

_______PROUINE 0 0 POLAR 0- _______CYSTEIE 0~ NONIONIZABLE ASPARAGINE 0- (POL) GLUTAMINE 0 ______TYROSINE 0 THREONINE 0 ______APARTIC ACID 0 IONIZABLE. ACIDIC 0mo ~Y S GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE: BASIC 2 CG YES___ TAG-ININE 2 POSITIVE CHARGE AGO YS HSDI.E(POS) I CODON 0 STOP SIGNAL 0 TTA T 4 3 Amino Acids Are Represented NPL: POL: NEG: POS. STP_ 1 T 2: 0: 0: 2: 0 TABLE 27. Mutagenic Cassette: N, G, T CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GOT YES GLYCINE I NONPOLAR _______ALANINE 0(NPL) VALINE 0 _______LEUCINE 0- 0 METHIONINE 0 ________PHENYLALANINE 0 _______TRYPTOPHAN 0 0 ATYES ISERINE I POLAR 2 TGT YES CYSTEINE I NONIONIZABLE SASPARAGINE 0 (POL) JGLIJTAMINE 0 JTYROSINE 0 ITHREONINE 0 SASPARTIC ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID- NEGATIVE CHARGE ______LYSINE 0 IONIZABLE: BASICI COT YES ARGININE I POsmTVE CHARGE IHISTIDINE 0(POS) STOP CODON 0 STOP SIGNAL 0 TOTAL 4 j4 Amino Acids r Represented NPL: POL: NEC: POS: STP I I 1: 2: 0: 1: 0 100 TABLE 28. Mutagenic Cassette: N, T, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTA YES VALINE I TIA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE I METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUITAMIC ACID 0 NEGATIVE CHARGE

INEG)

LYSINE 0 IONIZABLE BASIC 0 ARGININE POSITIVE CHARGE HISTIDINE 0 (POS) hTOPCODON 0 STOP SIGNAL 0

(STP)

TOTAL 4' 3 Amino Acids Are Represented NPL: POL: NEG: POS: STP- 1 0: 0: 0: 0 TABLE 29. Mutagenic Cassette: N, T, C CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE o (NPL) GTC YES VALINE I CTC YES LEUCINE I ATC YES ISOLEUCINE I METHIONINE 0 TIC YES PHENYLALANL4E I TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE o NONIONIZABLE ASPARAGINE o (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE BASIC 0 ARGININE POSITIVE CHARGE HISTIDNIE 0 (POS) STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0 TABLE 30. Mutagenic Cassette: N, T, G CODON Represented iAMINO ACID (Frequency) CATEGORY (Frequency) !LYCINE 0NONPOLAR 4 0 (NPL) GTG 'E VAUINEI rro YES LEUCINE 2 CTG YES _______ISOLEUCINE 0 ATG YES METHIONINE

I-

____________PHENYLALANINE 0 _______TRYPTOPHAN 0- 0- ______SERINE 0POLAR 0 05 NONIONIZABLE __________ASPARAGINE 0 (POL) GLUTAMINE0 _______TYROSINE 0 _______THREONIN 0 _________ASPARTI1C ACID7 0 IONIZABLE: ACIDIC 0.

GLUTAMIC ACID 0 NEGATIVE CHARGE I a

O'EG)

U0 IONIZABLE. BASIC o ARGININE 0 POSITIVE CHARGE 0 (POS) STOP CODON Are STOP SIGNAL 0

(STP)

TOTAL j 4 3 Amino AisreRepresented NPL: POL: NEG: POS: STP- 4: 0: 0: 0: 0 TABLE 31. Mutagernc Cassette: N, T, T CODON Represented AMINO ACID (frequency) CATEGORY (Frequency) _____GLYCINE 0 NONPOLAR 4 _______ALANINE 0 (NPL) GTT YES VALINEI CT!' YES LEUCINE

I

ATT YES ISO-LEUCINEI _______METHIONINE 0 TTT YES TPHENYLALM'JINE

I-

I_ TRYPTOPHAN 0 tPROLINE 0 SERINE 0POLAR 0 ______CYSTEINE 0 NONIONIZABLE A APARAGINE 0 (POL) _______GLUTAMINE 0 ________TYROSINE 0 ______THREONINE 0 tASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

_______ILYSINE U IONIZABLE: BASIC 0 T________ARGININE 0 POSITIVE CHARGE TOTA HISTIDINE 0 OS)A :0 TOAL4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP 102 00 TABLE 32. Mutagenic Cassette: N, A/C, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) wmmmw I GLYCINE 0 NONPOLAR. 2 GCA YES ALANINE I -(NPL) VAUINE 0 LEUCINE 0 ISOLEUCINE 0 METrHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCA YES PROLINE I TCA YES I -SERUNE I POLAR3 JCYSTEINE

NONIONIZABLE

SPARAGINE 0 (POL) CAA YES GLUTAMINE I- _______TYROSINE 0 ACA YES ITHREONINEI ASPARTIC ACID 0 IONIZABLE: ACIDIC I GAA YS GLUTAMIC ACID I NEGATIVE CHARGE

(NEG)

AAA YS LYSINE I IONIZABLE BASIC I ARGIN[NE 0~ POSITIVE CHARGE HISTIDINE 0(POS) 1A E STOP CODON I STIOP SIGNAL TOTAL 7 Amino Acids Are Represented NPL. POL: NEG: P05: STP- TABLE 33. Mutagenic Cassette: N, M/G, CODON Represented j AMINO ACID, (Frequency) CATEGORY (Frequency) GGA YES fGLYCINE I NONPOLARI ALANINE 0 (NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 MEMIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 0I POLAR I I CYSTEINE 0 NONIONIZABLE IASPAPLAGINE 0 (POL) CAA YES I GLUTAMINE I TYROSINE 0 THREONINE 0 _______ASPARTIC ACID 0 IONIZABLE- ACIDIC I GAR YES GLUTAMIC ACID NEGATIVE CHARGE

_(NEG)

AAA YES LYSINE I IONI.ZABLE: BASIC 3 CGA YES IARGININE 2 POSITIVE CHARGE AGA J YES

(POS)

HISTIDINE 0 TAR YES STOP CODON 2 STOP SIGNAL 2 TOA YES I(STW) TOTAL 8 5 Amino Acids Are Represented NPL: POL: NEG: POS: STP .1 ni J 1: 1: 3: 2 103 TABLE 34. Mutagenic Cassette: N, AJT, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) 0 NONPOLAR 4 ______ALANINE 0 (NPL.) GTA YES VALINEI TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINEI _______METHIONINE 0 0 _______TRYPTOPHAN 0 PROLINE 0 0 POLARI 0 NONIONIZABLE 0 (POL) CAA YES GLUTAMINE I- _______TYROSINE 0 THREONINE 0 ______jASPARTIC ACID C) IONIZABLE: ACIDIC GAA YES TGWUTAMIC ACID I NEGATIVE CHARGE

(NEG)

AAA YES LYSINE I IONIZABLE: BASICI _______ARGININE 0 POSITIVE CHARGE I4ISTIDINE 0 TAA YES STOP CODON I STOP SIGNAL TOTAL 8 6 Amino Acids Are Represented NPL: POL: NEG: POS: STP 4: 1: 1 1: 1 TABLE 35. Mutagenic Cassette: N, CIG, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGA YES GLYCINE I NONPOLAR 3 GCA YES ALANINE I (NFL) 0 0 SOLEUCINE 0 _______METHIONINE 0 0 0 CCA YES PROLINEI TCA YES SERUNE I POLAR 2 _______CYSTEINE 0 NONIONIZABLE ______ASPARAGINE 0 (POLW _______GLUTAMINE 0 ______TYROSINE 0 ACA YES THREONINE I- ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE-- BASIC 2 CGA YE-S ARGININE 2 POSITIVE CHARGE AGA YE(POS) HISTIDINE 0 TGA YES ISTOP CODON I STOP SIGNALI I (STW) TOTAL 8 1'6 Amino Acids Are Represented NPL: POL: NEG: POS: STP 1 3: 2: 0: 2: 1 104 TABLE 36._Mutagenic Cassette: N, CIT, A CODON Represented iAMINO ACID (Frequency) CATEGORY (Frequency) GLYIN E 0 NONPOLAR 6 GCA' YES ALANINE I (NPL) OTA YES VALINEI TA YES LEUCINE 2 CTA YES ATA YES ISOLEU-CINE I _______METHIONINE 0 0 0 CCA YES PROLINE I TCA YES SERINE I POLAR 2 ______CYSTEINE NONIONIZABLE ________ASPARAGINE 0 -(POL) GLUTAMINE 0 ______TYROSINE 0 ACA YES TIIREONINE I ________ASPARTIC ACID 0 1 IONIZABLE: AwmCIIC 0 TGLUTAMIC ACID 0 NEGATIVE CHARGE

MEG)

_____LYSINE 0 IONIZABLE: BASIC 0 0 STOP CODON 0i STOP SIGNAL .0 ____STm TOTAL 8 7 Amino Acids Are Represented Nit: POL. NEG: POS: STP-- 6: 2: 0: 0: 0 TABLE 37. Mutagenic Cassette: N, T/G, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGA YES GLYCINE I NONPOLAR ALANINE 0 (NPL) GTA YES VAUINEI iTA YES LEUCINE 2 CTA -YES ATA YES ISOLEUCINE I ______METHIONINE 0 PHENYLALANINE 0 0 0 ______SERINE 0 POLAR 0 0 NONIONIZABLE __________ASPARAGINE 0(POW) 0 _______TYROSINE 0 THREONINE 0 ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 2 CGA T YES ARGININE 2 POSITIVE CHARGE ES HISTIDINE (PS TGA j YES STOP CODON I STOP SIGNALI TOTAL 8 5 Amino Acids Are Represented NPL: IPOL: NEG: POS: STP 1- 5: 0: 0: 2: 1 105 TABLE 38. Mutagenic Cassette: N, C/ITf, A

CODUN

Rtepresented I AM1INO ACID (k requency) CATEGORUY (F.requency) u-I. 1 OA YES GLYCINE GCA YES ALANINE I- GTA YES VAUINEI TIA YES LEUCINE 2 CTAYE ATA YES ISOLEUCINEI METHIONINE 0

NONPULAR

(NPL)

7

YES

TRYPTOPHAN -0 PR2LNE I TCA YES SERINE

CYS'

ASF

GLU

0 E 0 0

NONIONIZABLE

(POL)

I YKUbINf: ALA V ~HKkONINh IASPARTIC ACID 0 ION IZABLE.- ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE I mFG ______LYSINE 0 IONIZABLE. BASIC 2 CGA YES JARGININE 2 POSrrM CHARGE AGA YES 1

(FOS)

THISTIDINE 0 TGA 1 YES 7STOP CODON I STOP SIGNAL I 4 (STW) TOTAL 12 I9 Amino Acids Are Represented NPL: POL: NEG: POS: STP I4__ 7: 2: 0: 2:1 TABLE 39. Mutagemc Cassette: N, A/Grr, CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGA YES IGLYCINE I NONPOLAR _______JALANINE 0 (NFL) GTA YES VALINE I TTA YES JLEUCINE 2 CTA YES ATA YES IISOLEUCINEI SMETHIO-41NE 0 PHENYLALANiNE 0 TRYPTOPHAN 0 PROLINE 0 SERINE. 0 POLARI _______CYSTEINE 0 NONIONIZABLE _______ASPARAGINE 0(POL) CAR YES GLUTAMINEI TYROSINE0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC I GAA YES GLUTAMIC ACID I~ NEGATIVE CHARGE

(NEG)

AAA LYSINE, I IONIZABLE: BASIC 3 CGA YESj ARG [NINE' 2 POSITIVE CHARGE AGA

(POS)

LHISTIDINE 0 TAR YES JSTOP CODO.N 2 STOP SIGNAL 2 TGA YS

(STP)

TOTAL 8 Amino Acids Are Represented NPL: POL: NEG: P05: STP 106 *TABLE 40. Mutagenic Cassette: N, Al/T, A CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) 0 NONPOLAR 6 GCA YES r ALANINE (NFL) GTA YES VALINEI TAYES LEUCINE 2 CIA YS ATA YES ISOLEUCINE I METHIONINE 0 PHENYLALANINE 0 0 CCA YES I[PROLINEI TCA YES I SERINE I POLAR 3 JCYSTEINE 0 NONIONIZABLE JASPAKAGINE 0 (POL) CAA YES JGLUTAMINEI TYROSINE 0 ACA YES ITHREONINE I- IASPARTIC ACID_ 0 IONIZABLE.~ ACIDIC I GAA YES TGLUTAMIC ACID I NEGATIVE CHARGE _I (EG) AAA YES LYSINE I IONIZABLE. BASIC I IARGININE 0 POSITVE CHARGE UHISTIDINE 0 (POS) TAA J YES ISTOP CODON I STOP SIGNALI

(STW)

TOTAL 12 I10 Amino Acids Are Represented NPL: POL: NEG: POS: STP U 6: 3: 1: 1 I TABLE 41. Mutagenic Cassette: N, AJCIG, CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GGA YES GLYCINE I NONPOLAR 3 GCA YES ALANINE I (NPL) VAUINE 0 LEUCINE 0 ISOLEUCTNE 0 METHIONINE 0 PHENYLALANINE 0 ______TRYPTOPHAN 0 CCA YES IPROLINE I- TCA YES SERINE I POLAR 3 CYSTEINE 0 NONIONIZABLE _______ASPARAG[NE 0 (POL) CAA YES jGLUTAMINEI TYROSINE 0 ACA YES THREONINE I ACID 0 IONIZABLE.~ ACIDIC I OAR YES jGLUTAMIC ACID I NEGATIVE CHARGE

(NEG)

AAA YES LYSINE I IONIZABLE: BASIC 3 CGA YES [ARGIN1I4E 2 POSITIVE CHARGE AGA YES FHISTIDINE 0 TA ES ISTOP CODON 2 STOP SIGNAL2 TGA YES_ I_ TOTAL 12 I9 Amino Acids Are Represented NPL: POL: NEG: POS: STIP jI3: 3: I1 3: 2 107 TABLE 42. Mutagenic Cassette: A, N, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR. 4 ALANINE 0 (I4PL) VALINE 0 LEUCINE 0 ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINEI ACT YES THRENINE 4 ACA Y ES SRN OA ASPRTI ACM UIONINABE:ACIDC CYSTENE 0(NEG) AAA YES LSINAE 2 INZALBSC4 AATYOSN PO0ECAG ACT YES ARININE 4 AC YES SARI 4:NIABL: 0:ID 4: 0 CODON Repreented AMINO ACID (Feuny CEATIEGORY Frqen AAA YES PLYINE 4 OIAL&AI CCC YESf

OIIECAG

AGA YES AGNN

PS

AGO YES KSRINE 0POA2 CYSTENE o ONIONZABL TALA3 uaei asetAe:ACN, NPL CAR YES 0GLOTAMINR 2 TYROINE 0 NL THREINE 0 I F YES LECIE COC YES CO YES CAT YES PROSTIINE 4 CAC YES 1 1 STOP ODO 0 PTOLIGA 0 _N 0 NONOTPABL TOTAL 16 5 AmiAIdsAr Rersne NP OL: E:PS T CAA8 2ES 0:FF N 6:2

-IG

TYROSINE8 TABLE 44. Mutagenic Cassette: G, N, N CODON I Represented I AhUN0 A D (Frequency) I CATEGORY (Frequency) F S S NONPOLAR GG1 czr

GLYCINE

(NPL) YES CK00 YES GCT YES ALANINE 4 GOC YES GCA YES GCG YES OTT YES VALINE 4 GTC YES GTA YES GTG YES LEUICINE0 POLAR 0)

NONIONIZABLE

(POL)

0 1TYROSINE

ITHREONINE

GAT YES I ASPARTICA WllLA LIJ 0 NEGATIVE CHARGE

(NEG)

GLUTAMIC ACID 2 I

I

IlfOIZALE BASICt 0 POSITIVE CHARGE TOTA 16 5 Anuo cidsAreRepresented NPL: POL: NEG: POS: STP- 12: 0: 4: 0: TABLE 45. Mutagenic Cassette: T, N, N CODjON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GYIE0NONPOLAR 3 ALANNE 0(NPL) Trc YES MM TGG YES TYTPA PROLEINE 0 TCT YES SENELLNN 2 OA TC YES(PL TGT YES CYT N I APRONE 0 GLUTYSAMEINE 4 OA0 TAT YES TYROSINE 2

TACYE

THREONINE 0 ASPRI CD IONIZABLE- ACIDIC LI 1 GLUTIC ACID 0 NEGATIVE CHARGE ACD

NEGI

LYSINE 0 IONIZABLE, BASIC 0 ARGININE 0POSITIVE CHARGE JI IIISTIDINE 0 (POS) TAA YS jSTOP CODON 3 STOP SIGNAL 3 TOTAL I 16 6 Amino Acids Are Represented NPL: POL: NEG: P05: STP 8: 0: 0: 3 109 *TABLE 46. Mutagenic Cassette:'A/C, N, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) _____GLYCINE 0 NONPOLAR 12 _____ALANINE 0 (NPL) ______VALINE 0 CTT YES LEUCINE 4 CTC YES CTA YES CTG YES All' YES ISOLEUCINE 3 ATC YES -ATA YES ATG YES METrHIONINE

I

PHENYLALANINE 0 ______TRYPTOP-AN 0 CCT YES PROLINE 4 CCC YES CCA YES CCG YES AGT YES SERENE 2 POLAR AGC YES

NONIONIZABLE

______CYSTEINE 0 (POL) AAT YES ASPARAGINE 2 AAC YES CAA YES GLUJTAMINE 2 CAG YES 0 ACT YES THREONINE 4 ACC YES ACA YES I ACG YES IASPARTIC ACID 0 IONIZABLE: 0 GLUTAMIC ACID 0 ACIDIC NEGA77VE

CHARGE

AAA YES I LYSINE 2 IONIZABLE. BASIC AAG YES

CHARGE

CGT YES ARGININE 6 (POS) CGC YES CGA YES CGG YES AGA YES AGG YES CAT YES HISTIDINE 2 CAC YES STOP CODON 0 STOP SIGNAL 0 TOTAL 32 11 Amino Acids Are Represented NLPL:NEG: POS: STP 1 12: 10: 0: 10: 0 110 TABLE 47. Mutagenic Cassette: A/G, N, N CODON Represented AMINO ACID (frequency) CATEGORY (Frequency) GOT YES GLYCINE 4 NONPOLAR 16 GGC YES

(NPL)

GGA YES GGG YES GCT YES ALANINE 4 GCC YES OCA YES GCG YES GT YES VALINE 4 GTC YES GTA YES GTG YES LEUCINE 0 ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE I PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 AGT YES SERINE 2 POLAR 8 AGC YES NONIONIZABLE CYSTENE 0 (POL) AAT YES ASPARAGINE 2 AAC YES GLUTAMINE 0 TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 4 AAG YES POSITIVE CHARGE AGA YES ARGININE 2 (POS) AGG YES HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 32 12 Amino Adds Are Represented NPL: POL: NEG: POS: STP- 16: 8: 4: 4: 0 111 TABLE 48. Mutagenic Cassette: AfT, N, N CODON IRepmented IAMINO ACID lFrequcocyl CATEGORY (Frequeucy) 0 NONPOLAR 9 0 (NPL) 0 TrA YES LEUCINE 2 TTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE I M~ YES PHENYLALANINE 2 TTC YES TOG YES TRYPTOPHANI PROLINE 0 TCT YES SERINE 6 POLAR 16 TCC YES NONIONIZABLE TCYES (POL) TCG YES AGT YES AGC YE-S TOT YES CYSTEINE 2

TUCYE

AAT YES ASPARAGINE 2 AAC YES _______GLUTAMINE 0 TTYES TYROSINE 2 TAC YES ACT YES THREONINE 4 ACC YES ACA YES ACO YES ASPARTIC ACID 0 IONIZABLEACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE AA I YES LYSINE 2 IONIZABLE. BASIC 4 AAG i: YES POSITIVE CHARGE AGA YES ARGININE 2 t STlDfNE 0 TAR YES STOP CODON 3 STOP SIGNAL 3 TAG j YES

TP

TOTAL I 32 12 Amino Acids Are NPL: POL: NEG: POS: STP= IRepresented 9: 16: 0: 4: 3 112 TABLE 49. Mutagenic Cassette: CIG, N, N CODON Bpmmend AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR GGC YES (NL) GGA YES GGG YES GCT YES ALANINE 4 0CC YES GCA YES GCO YES GT YES VALINE 4 GTC YES OTA YES GTG YES CIT YES LEUCINE 4 CTC YES CrA YES CTG YES ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES CCG YES SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAOINE 0 (POL) CAA YES OLUTAMINE 2 CAG YES TYROSINE 0 THREONINE 0 OAT YES ASPARTIC ACID 2 IONIZABLE. ACIDIC 4 GAC YES NEGAIVE CHARGE OAA YES OLUTAMIC ACID 2 (NEG)

YES

LYSINE 0 IONIZABLE- BASIC 6 CGT YES AROININE 4 POSITIVE CHARGE COC YES CGA YES COO YES CAT YES HISTIDINE 2 CAC YES STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 32 10 Amino Acids Are NPL: POL: NEC: POS: STP R epresented 20: 2: 4: 6: 0 TABLE 50. Mutagenic Cassette: C/T. N, N CODON Represented IAMINO ACID (Frequency) CATEGORY (Frequency) LYCINE 0 NONPOLAR 13 _______ALANINE 0 (NPL) 0 TTA YES LEMIINE 6 CTC YES CTA YES Cra YES ________ISOLEUCINE 0 ________METHIONINE 0 TITr YES PHENYLALANINE 2 TTC YES TOG YES TRYPTOPHAN

I

CCT YES PROLINE 4 CCC YES CCA YES CCO YES TCT YES SERINE 4 POLAR TCC -VE-

NONIONIZABLE

TCA YES

(POL)

TCG YES TOT YES CYSTEINE 2 TOC YES ________ASPARAGINE 0 CAA YES GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES THREONINE 0 ______ASPARTIC ACID 0 IONIZABLE. ACIDIC 0 OLUTAMIC ACID 0 NEGATIVE CHARGE i. -G LSN 0 IONIZABLE- BASIC 6 COT YES ARGININE 4 PSMVE CHARGE CGC YES

(POS)

COA YES COG YES CAT YES HISTIDINE 2 TAR YE STO P CODON 3 STOP SIGNAL3 TAG YES j(S?) TOTL 32 10 Amino Acids Are NPL: POL: NEG: POS: STP Represented 13: 10: 0: 6: 3 114 TABLE 51. Mutagenic Cassette: GIT, N, N CODO N Repreented AMINO ACID (Frequeneyp CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 1 GOC YE-S (NPL) GGA YES 000 YES OCT YES ALANINE 4 0CC YES UCA YES GCO YES OTT YES VALINE 4 GTC YES GTA YES GTO YES iTrA YES LEUCINE 2 _________ISOLEUCINE 0 _______METHIONINE 0 i-IT YES PHENYLALANINE 2 TCYES TOO YES TRYPTOPHAN I PROLINE 0 TCT YES SERINE 4 POLAR 8 TCC -yTSNONIONIZABLE TCA YES (POL) TCG YES TOT YES CYSTEINE 2 TGC ________ASPARAGINE 0 LUTAMINE 0 TAT YES TYROSINE 2 TAC Y-E-S THREONINE 0 GAT YES ASPARTIC ACID 2 IONLZABLE. ACIDIC 4 GAC YE NEGATIVE CHARGE OAR YES OLUTAMIC ACID 2 (NE) GAG YES 0 IONLZABLE. BASIC 0 0 P'OSITIVE CHARGE HISTIDINE 0 OS TAR YES STOP CODON 3 STOP SIGNAL 3 TAG YES SP TOA YES TOTAL 32 11 Amino Acids Are NFL: POL: NEG: POS: STP j Represented 17: 8: 4: 0: 3 115 TABLE 52. Mutagenic Cassette: N, A, N mm mm I MqftsE~~f~ CATEGORY rfI(c

NONPOLAR

(NPL)

VALINE

L

LEUINE

-ISOLEIJC

METHIOI

-b.i

SINE

POLAR

NONIONIZABLE

(POL)

AAl

ARC

YS ASPAPAGINE CAA YS GLI)AMINE -2

TAT

YES

y F-9 OAT I YES -AC I -vWq THRE-ONINE0 IASPARTIC Armi GAA YES

GAG

GLUTAMIC ACID IIZnABLE: ALADIC NEGATIVE CHARGE

(NEC)

AAA YES LYSINE 2IONIZABLE: BASIC 4 AAGRTf POSITIVE CHARGE aARG11NINE 0 (POS) CT YES HISTIDINE 2 CAC I ES TAR YES -STOP CODON 2 STOP SIGNAL2 TAG YES

SP

TOA 6 7 Amino Acids Are Represented NFL: POL: NEG: POS: STP 0: 6: 4: 4: 2 TABLE 53. Mutagenic Cassette: N, C, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) I LICINE 0NNOA OCT ES LANIE 4(PL) AC

YES

VALEG)

LYSINE0 0 O I A L A I ARGILNINE 0 OmECAG SRTOP O N 0 TPSINL( TOT 4E AmROino Acd4r ersne F:PL E:P T 1o 8 8 0 116 TABLE 54. Mutagenic Cassette: N, G, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GTYS -GYIE4 NONPOLAR GGC YES(NPL) ALANINE 0 VALINE 0

LEUCINE

ISOLEUCINE 0 METHIONINE I) PHENYLALANINE 0 TOG YES TRYPTOPHANI PROLINE 0 AG E EIE2 POLAR 4 AGC YESNONION17A LE TGT ES CSTEIE 2(POL) ASARAGINE 0 GLIUTAMINE 0 TYROSINE 0 THREONINE 0 ACD IONIZAB).E: ACIDIC GLWTAMC ACD 0 NEGATIVE CHARGE LYSINE a) IONIZABLE. BASIC CG ES AGIIE6 POSIIVE CHARGE CGC YES (POS) CGA YES AGA S AGG YES HISTIDINE 0 TGA YES STOP CODON I STOP SIGNAL _(SmP TOAL16 5 Amino Acds Are Represented NPL: POL: NEG: POS: STP r -OT5: 4: 0: 6:1 TABLE 55. Mutagenhc Cassette: N, T, N CODm I -tpme -I Raprmt~ Irequefacy) CATEGORY (FreIcuv ALANINE 0 NL OTT YES VALINE 4 GTC YES GTA YES GTG YES YA YES LEUaINE 6

CIC

NONPOLAR

(NPL)

0 POLAR 4) 2

NONIONIZABLE

(POLl

ASPARAGINE

~GLUTAMINE

IONIZABLE: ACIDIC NEGATIVE CHARGE

(NEGI

0 I 0 IONIZABLE. BASIC 0 POSITVE CHARGE _0 H

STOPCODON

(STm 0 TOTAL 16 r AioAisAeRpseed NFL: POL: NEG: P05: STPmn Acdjr ersne 16: 0: 0: 0: 0 117 TABLE 56. Mutagenic Cassette: N, A/C, N CODON "07Represented AMINO ACID (Frquency) CATEGORY (Frequency) ______GLYCINE 0 NONPOLAR GCT YES ALANINE 4 (NPL) GCC YES GCA YES GCG YES I 0 _______LEUCINE 0 ________ISOLEUCINE 0 _______METHIONINE 0 PHENYLALANINE 0 ________TRYPTOPHAN 0 CC-r YES PROUINE 4 CCC YES CCA YES CCG YES TCT YES SERINE 4 POLAR 14 TCC YES

NONIONIZABLE

TCA YES

(POL)

TCG I YES _______CYSTEINE 0 AAT YES ASPARAGINEI AAC YES CAA YES GLUTAMINE2 CAG YES TAT YES TYROSINE TAC YES ACT YES THREONINE 4 ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2 IONIZABLE. ACIDIC 4 GAC YES NEGA77VE CHARGE GAA YES GLUTAMIC ACID 2 (NE(3) -GAG YES AAYES LYSINE 2 IONIZABLE: BASIC 4 AAC YES POSITVE CHARGE _______ARGININE 0 CAT YES HISTIDINE2 TAAL Y ES ITP CODON 2 STOP SIGNAL2 TAG I YES ~SO(STP) TOT11 Amino Acids Areresented NPL: POL: NEG: POS: STP 8: 14: 4: 4:2 118 TABLE 57. Mutagenic Cassette: N, A/G, N CODON j Repmented AMINO ACID (Frequency) CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR GGC YES (NPL) GGA YES ALANINE 0 VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGO YES TRYPTOPHAN I PROLINE 0 AGT YES SERINE 2 POLAR AGC YES NONIONIZABLE TOT YES CYSTEINE 2 (POL) TGC YES AAT YES ASPARAGINE 2 AAC YES CAA YES GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES THREONINE 0 OAT YES ASPARTIC ACID 2 IONIZABLE ACIDIC 4 GAC YES NEGATIVE CHARGE GAR YES GLUTAMIC ACID 2 (INEG) GAG YES AAA YES LYSINE 2 IONIZABLE BASIC (0 AAG YES POSITIVE CHARGE COT YES ARGININE 6 (POS) CGC YES CGO COA YES AGA YES AGG YES CAT YES THISTIDINE 2 CAC YES TAR YES I STOP CODON 3 STOP SIGNAL 3 TAG YES (Si) TGA YES TOTAL 32 12 Amino Acids Are Represented NPL: POU NEG: POS: STP _I 1 1 5: 10: 4: 10: 3 119 TABLE 58. Mutagenic Cassette: N, AJT. N CODON Represented rCATEGORY (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 16 J ALANINE o (NPL) GIT YES VALINE 4 GTC YES GTA YES GTG YES TrA YES LEUCINE 6 0TG YES 00CT

YES

CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 SATC YES In I ATA YES ATG YES METHIONINE

I

iTr YES PHENYLALANINE I YES TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR CYSTEINE o NONIONIZABLE AAT YES ASPARAGINE 2 (POL) AAC YES CAA YES GLUTAMINE CAG YES TAT YES TYROSINE 2 TAC YES THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES LYSINE 2 IONIZABLE.- BASIC 4 AAG YES POSITIVE CHARGE ARGININE 0 (POS) CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 2 STOP SIGNAL 2 TAG YES (SP) TOTAL 32 12 AmIno Adds Are Represented NPL: POL: NEG: POS: STP 1 _16: 6: 4: 4: 2 120 TABLE 59. Mutagenic Cassette: N, CIG, N CODON Represented AMINO ACID (Freq-ey) CATEGORY (Frequency) GOT YES GLYCINE 4 NONPOLAR 13 GGC YES (NPL) GGA YES 000 YES OCT YES ALANINE 4 0CC YES OCA YES GCG YES 0 0 _______ISOLEUCINE 0 _______METHIONINE 0 ________PHENYLALANINE 0 TGG YES TRYPTOPHAN I- CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT I YES SERINE 6 POLAR 12 TCC YES NONIONEZABLE TCA YES (POL) TCU YES ACUT YES AGC YES TOT YES CYSTEINE 2 _______ASPARAGINE 0 _______GLUTAMINE 0 ______TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

_____LYSINE 0 IONIZABLE: BASIC 6 COT YES AROININE, 6 POSITIVE CHARGE CGC YS(POS) COA YES

YE-

AGA YES AGO YES IHISTIDINE 0 TGA YES STOP CODON I STOP SIGNALI 32 8 Amnino Ad~ds Are Represented NPL: POL: -NEG: POS: STP i l 13: 12: 0: 6: 1 121 TABLE 60. Mutagenic Cassette: N, C/T. N coorI CODON Represeuted AMINO ACID (frequency) CATEGORY lFrequtocy) GLYCINE 0 NONPOLAR 24 GCT YES ALANINE 4 '(NPL) GCC YES GCA YES GCG YES GTT YES VAUNE 4 GTC YES GTA YES GTG YES TM YES LEUCINE 6 -TrO YES CIT YES CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE I lTm YES PHENYLALANINE TIC YES TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES SERINE 4 POLAR 8 TCC YES

NONIONIZABLE

TCA YES

(POL)

TCG YES CYSTEINE 0 ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC ACID 0 IONIZABLE- ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 0 ARGI NINE 0 POSITIVE CHARGE HISTIDINE 0 (POS), STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 32 9 Amino Adds Are Represented NFL: POL: NEG: P05: STP 24: 8: 0: 0: 0 122 TABLE 61. Mutagenic Cassette: N, GIT, N CODON Reprnted I AMINO ACID (Frequency) CATEGORY (Frequency) GT YES GLYCINE 4 NONPOLAR 21 GGC YES (NPL) GGA GGG YES I I ALANINE 0 'IT I YES VALINE 4 GTC YES GTA YES GTG YES TTA YES LEUCINE 6 ITO YES CTC YES CTA YES Cro YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE iTr YES PHENYLALANINE 2 ITC YES TGG YES TRYPTOPHAN PROLNE 0 AGT YES SERINE 2 POLAR 4 AGC YES NONIONIZABLE TGT YES CYSTEINE 2 (POL) TGC YES ASPARAGINE 0 _LUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 OLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE: BASIC 6 COT YES ARGININE 6 POSITIVE CHARGE YES (POS) CGA YES COO YES AGA YES AGO YES HISTIDINE 0 TGA YES T STOP CODON I STOP SIGNAL I

(STP)

S 32 10 Amino Adds Are Represented NPL: POL: NEG: POS: STP 21: 4: 0: 6: 1 123 TABLE 62. Mutagenic Cassette: N, AICIG, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GOT YES GLYCINE 4 NONPOLAR 13 GOC YES

(NPL)

OA- YES GGG YES 1 OCT YES IALANINE 4 GCC YES GCA YES GCG YES ________METHIONINE 0 TGG YES rTRYPTOPHAN

I

CCT YES PROLINE4 CCC -YES CCA YES CCG YES TCT YES SEIE 6 POLAR

IS

TCC YSNONiONIZABLE TCA YS(POL) TCO YES AGT YES AOC YES TOT YES CYSTEINE2 TGC YES ART YES ASPARAGINE2 ARC YES CAA YES GLUTAMINE 2 _CAG YES TAT YES TYROSINE TAC YES ACT YES THREONINE 4 ACC YES ACA YES AG YES OAT YES ASPART1C ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE OAR YES GLUTAMIC ACID 2 (NEG) GAOA- YES AAA YES LYSINE 2IONIZABLE: BASIC AROWG YES POSITIVE CHARGE COT YES ARGININE 6 (POS) M -C YES CW YES AGA YES AGO YES CAT YES HISTIDIE2 CAC YES TAR YES STOP CODON 3 STOP SIGNAL 3 TAG YES

(STW)

TGA YES L TOTAL 48 15 Amino Acids Are Represented NPL: POL: NEG: POS: STP 124 TABLE 63. Mutagenic Cassette: N. A/C/T, N CODON Represeted AMINO ACID ffrequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 24 OCT YES ALANINE 4 (NPL) 0CC YES OCA YES OCG YES GTr YES VAUINE 4 OTC YES GTA YES OTOj YES TTA YES LEUCINE 6 flU- YES CiT YES CTC YES CA YES CT YES AlT YES ISOLEUCI-NE 3 ATC YES ATA YES ATO YES METHIONINE

I

IT YES PHENYLALANINE 2 Trc YES ________TRYPTOPHAN 0 CCT YES PROUNE 4 CCC YES CCA YES CCO YES TCT YES SERtINE 4 POLAR 14 fCC YES

NONIONIZABLE

TC-A YES

(POL)

TCG -YES- CYSTEINE 0 ART YES ASPARAGINE 2

AACYE

CAA YES -GLUTAMINE 2 C G YES TAT YES TYROSINE -TAC YES ACT YES THREONINE 4 -ACC YES ACA YES ACG YES OAT YES ASPARTIC ACID' 2 IONIZABLE. ACIDIC 4 GAC NEGATIVE CHARGE GA YES GLUTAMIC ACID 2 (NWG) GAG" YES AAA ;YES LSINE 2 IONIZABLE: BASIC 4 AAG POSITIVE CHARGE _______ARGININE 0 CAT YES HISTIDINE 2 -CAC YES TAR YES STOP CODON 2 STOP SIGNAL 2 TAG YES

TP

TOTAL 41 16 Amino Adids Are Represente NPUj POL: NEG: POS: STP 24: 14: 4: 4: 2 TABLE 64. Mutagenic Cassette: N, AIGrr, N CODON Represented AMINO ACID (frequency) CATEGORY lFrequency) GGT YES GLYCINE 4 NONPOLAR 21 S YES

(NPL)

GGA YES 000 YES _______ALANINE 0 7T YES VALINE 4 OTC YES GTA YES GTG YES TTA YES LEUCINE 6 TFG YES CT YES CTC YES CTA YES CTG YES ART YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE I MT YES PHENYLALANINE 2 rC YES TOG YES TRYPTOPHAN

I

PROLINE 0 AGT YES SERINE 2 POLAR AGC YES

NONIONIZABLE

TGT YES CYSTEINE 2(POL) TGC -YE-S AAT YES ASPARAOINE2 AAC YES CAA YES GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUJTAMIC ACID 2 (NEG) G-A-G YES AM," YES LYSINE 2 IONIZABLE. BASIC AAO YES POSITIVE CHARGE COT YES ARGININE 6 CGC YES -CGA YES COG YES AGA YES A--GO YES CA ES HISTIDINE 2 -CAC YE TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES J(ST?) TGA YES TOTAL 48 j17 Amino Acids Are Represented NPL: POL: NEG: P05: STP 21: 10: 4: 10: 3 126 TABLE 65. Mutagenic Cassette: N, C/GIT, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) -GGT YES OLYCLNE 4 NONPOLAR 29 OGC YES (NFL) GOA YES 000 YES OCT YES ALANINE 4 0CC YES OCA YES GCG YES GTT YES VALINE 4 GTC YES OTA YES OTO YES TMA YES LEUCINE 6 TrG YES rr YES CTT YES CTA YES CTA YES ATF YES ISOLEUCINE 3 ATC YES ATA YES ATO YES METHIONINE I TrT YES PHENYLALANINE 2 TTC YES TOO YES TRYPTOPHAN I CCT YES PROLINE 4 CCC YES CCA YES CCO YES TCT YES SERINE 6 POLAR 12 TCC YES NONIONIZABLE TCA YES (POL) TCG YES AGT YE AGC YES TOT YES CYSTEINE 2 TGC YES ASPARAG[NE 0 _LUTAMINE 0 TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA Y ACO YE ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 OLUTAMIC AID 0 NEGATIVECHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 6 COT YES AROININE 6 POSITIVE CHARGE CGC -ycvf (POS) CGA YES CGO YES AA YES AGO YES HISTIDINE 0 TGA YES STOPCODON I STOP SIGNAL I

(STP)

TOTAL 48 13 Amino Adds Are Represented NFL: POL: NEG: POS: STP= 29: 12: 0: 6: 1 127 TABLE 66. Mutagenic Cassette: C, C, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0 LEUCINE 0 ________ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE POA0 CCT YES R(POL) GITMI ACI POALVECARGE YSTINE 0 NIONIZABLE AI AR G INE 0 POITVECHRG HITDINE 0~ TOP CON 0SOPIGA0 r TOTAL 4 LUTI in ACiID 0ersne NEGL:V CHARE:P T 0: 0OIAL: 0:S 0: 0 TABLEIN 67 MutSgeIVc Casete:RGE ~~~~TPCODON 0 RSrsue MN CD (rqec) CTO Y (FreqAency GGT YES GLYCINE 4 NONPOLAR4 GGC YES (NPL) GGA YES GGG f YES ALANINE 0 VALINE 0 LEUCINE 0, -ISOLEUCINE0 -METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 STEINE NONONIZA 0:0 HISTIDAINE 0 (POS) STYOSIN 0STPINA0 J TOTAL 0 4 j UTI i ACiIs Rersne NEGAIV CHLARE PS T LYSINE 0 IONIZABLE: BASIC 0 4: 0 128 TABLE 68. Mutagenic Cassette: G, C, N CODON IRepresented AMINO ACID (Frequency) V lFrequency) GLYCINE 0 GCT YES ALANINE4 GCC YES GCA YES GCG YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROUINE0

NONPOLAR

(NPL)

0 POLAR 0 -CYSTEINE 0 NONIONIZABLE ________ASPARAGINE 0 (POL) -GLUTAMINE 0 _______TYROSINE0 THREONINE0 _______ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE. BASIC 0 0 POSITIVE CHARGE HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 TOTAL I 4 1 Amino Add is Represented NPL: POL: NEG: POS: STP II4: 0: 0: 0:0 TABLE 69. Mutagenic Cassette: G, T, N CODON Represented AMINO ACID (Frequency). CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 _______ALANINE 0 (NFL) GTT YES VALINE 4 GTC YES GTA YES GTG YES LEUCINE 0 _______ISOLEUCINE 0 ________METHIONINE 0 PHENYLALANINE 0 ________TRYPTOPHAN 0 PROLINE 0 SHRINE 0 POLAR 0 ______CYSTEINE 0 NONIONIZABLE ______ASPARAGINE 0 (POL) _______GLUTAMINE 0 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE -HISTIDINE 0 OS ZSTOP CODON 0 STOP SIGNAL 0 TOTAL4 1 Amino Acid 1, Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0 129 TABLE 70. Mutagenic Cassette: C, G, N CODON Represented AMINO ACID) (Frequency) CATEGORY lFrequency) 0 NONPOLAR0 -ALANINE 0 (NPL) 0 0 ISOLEUCINE 0 _______METHIONINE 0 ________PHENYLALANINE 0 _________TRYPTOPHAN0 PROLINE0 0 POLAR 0 -CYSTEINE 0NONIONIZABLE ________ASPARAGINE 0 (POL) 0 0 THREONINE 0 _______ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

_____LYSINE 0) IONIZABLE-- BASIC 4 COT YES ARGININE 4 POSITIVE CHARGE CGA YES O) CG 1 YES C8 I YS HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 iWNTOTA 4 1 Amino Acdd Is Represented NPL: POL: NEG: POS: STP 0: 0: 4: 0 TABLE 71. Mutagenic Cassette: C, T, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequeny) 0 NONPOLAR 4 0 (NPL) VAL2NE 0 CiT YES LEIJCINE 4 CTC YES CTA YES CTO YES _______ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0- _______TRYPTOPHAN 0 PROLINE 0 0 POLAR 0 ______CYSTEINE NONIONIZABLE ______ASPARAGINE 0 -(POL) ______GLUTAMINE 0- ______TYROSINE 0 THREONINE 0- _____ASPARTIC ACID 0 IONIZABLE~ ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 TOTAL 4 1 Amino Acid Is Represented NFL: POL: NEG: POS: STP I4: 0: 0: 0: 0 130 TABLE 72. Mutagenic Cassette: T, C, N CODON IRepreented -1 AMINO ACID (Frequency) (Freqaency)

GLYCINE

ALANINE 0 VALINE0 LEUCINE0 _______ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0

(NPL)

TCT YES SERINE 4 POLAR 4 TCC YVES NONION17ABLE TCA YES (POL) TCG YES 0 _______ASPARAGINE 0 _______GLUTAMINE 0 0 THREONINE0 ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE: BASIC 0 RGININE 0 POSTVE CHARGE TOTAL LSTINE 0 (P05) PS T 0: 4: 0: 0: 0 TABLE 73. Mutagenic Cassette: A, C, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) OLYCINE 0 NONPOLAR 0.

_______ALANINE 0 (NPL) 0 0

_______ISOLEUCINE

_______METHIONINE 0 ________PHEYLALANINE 0 0 PROLINE 0 0 POLAR 4 _______CYSTEINE 0 NONIONIZABLE _______ASPARAGINE 0 (POL) _______GLUTAMINE 0 ______TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES _____ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE z n LYSINE 0 IOIZB.BASIC 0 0 PSTVCHARGE STOP CODON 0 STOP SIGNAL 0 TOTAL 4 '7 Amino Acid Is Represented NFL: POL: NEG: POS: STP 0: 4: 0: 0: 0 131 *TABLE 74. Mutagenic Cassette: G, A, N CODON Represented AMINO ACID fFrequency) CATEGORY lFrequency) _____GLYCINE 0 NONPOLAR 0 _______ALANINE 0(NFL) 0 ILEUCINE 0 IISLEUCINE 0 0 ________PHENYLALANINE 0 _______TRYPTOPHAN 0 PROLINE0 ______SERINE 0 POLAR 0 _______CYSTEINE 0 NONIONIZABLE ________ASPARAGINE 0 (POL) _______GLUTAMINE 0 0 THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES 0 IONIZABLE: BASIC 0 ARGININE POSITIVE CHARGE IHISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 TOTAL 4 2 Amino Acids Are Represented NPL: POL; NEG: P05: SIP 0: 4: 0: 0 TABLE 75. Mutagenic Cassette: A, T, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 4 _______ALANINE 0 (NPL) 0 07 A YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METIONINEI IHENYLALANINE 0 ______TRYPTOPHAN 0 PROLINE 0 _____SERINE 0 POLAR 0 0 NONIONIZABLE 0(POL) ______GLUTAMINE 0 _______TYROSINE 0 THREONINE 0 ACID 0 IONIZABLE:~ ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG3) ______LYSINE 0 IONIZABLE- BASIC 0 _______ARGININE 0 POSITIVE CHARGE HISTIDINE 0(POS) STOP CODON 0 STOP SIGNAL 0 TOTAL 4 2 Amino Adds Are Represented NPL: POL: NEG: P05: STP= 4: 0: 0: 0: 0 132 TABLE 76. Mutagenic Cassette: C, A, N CODON Representea IAMINO ACID (Frequenc) 1 LLYCINE 0 ~ALANINE 0NL lFreqUency)

NONPOLAR

VALINE

0 0 E 0 dJINE 0 PHENYLAL S ERINE PHAN 0 0 NE 0 IE 2

POLAR

NONIONIZABLE

(POL)

L AAj I AQ YES

ULUTAMIN

ml-TYROSIN~E0 ________ASPARTIC ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE. BASIC2 ________ARGDN1NE 0 POSITIVE CHARGE CAT YES HLITIDINE 2 (POS) CAC YES STOP CODON 0 STOP SIGNAL 0 TOTAL 4 2 Amino Acids Are Represented NFL: POL: NEG: POS: STP I1 0: 2: 0: 2: 0 TABLE 77. Mutagenic Cassette: T, T, N CODON rRepresented AMINO ACID (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 4 0 (NPL) 0 TTA YES LEUCINE 2 TTG YES 0 _______METHIONINE 0 Mf YES PHENYLALANINE 2 IrC YES _________TRYPTOPIIAN 0 PROLINE 0 _____SERINE 0 POLAR 0 _______CYSTEINE 0 NONIONIZABLE 0 (POL) 0 0 THREONINE 0 ACID 0 IONIZABLE: ACIDIC 0 GLUTANIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE. BASIC 0 0 POSITVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 TOTAL 4 2 Amino Acids Are Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0 133 TABLE 78. Mutagenic Cassette: A, A, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 0 0 (NPL) 0 LEUCINE 0 ISOLEUCINE 0 ________METHIONINE 0 PHENYLALANINE 0 ________TRYPTOPHAN 0 ______PROLINE, 0 0 POLAR2 ________CYSTEINE 0 NONIONIZABLE ASPARAGINE. 2(OL AAC YES GLUTAMINE 0 _______TYROSINE 0 _______THREONINE 0 _______ASPARTIC ACID 0 IONIZABLE.* ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG).

AAA YES LYSINE 2 IONIZABLE: BASIC 2 AAG YES POSITIVE CHARGE ARGININE 0 (POS) THISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 TOTAL j 4 12 Amino Acids Are Represented NPL: POL: NEG: POS: STP= J 0: 2: 0: 2: 0 TABLE 79. Mutagenic Cassette: T, A, N CODON RePpresented AMINO ACID (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 0 ALANINE 0 -(NPL) 0 ________ISOLEUCINE 0 ________METHIONINE 0- _________PHENYLALNINE 0 ________TRYPTOPHAN0 PROLINE 0 SERENE 0POLAR 2 _______CYSTEINE 0NONIONIZABLE ASPARAGINE

(POLW

GLLITAMINE0 TAT YES TYROSINE2 TAC YES _______THREONINE 0 ASPARTIC ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE 0 IONIZABLE. BASIC 0 ARGININE 0 POSITVE CHARGE, -HISTIDINE 0(lOS) TARA YES STOP CODON 2 STOP SIGNAL 2 TAG YES

(STW)

TOTAL 4 1 Amino Add Is Represented NPL: POL: NEG: POS; STP 0: 2: 0: 0: 2 134 TABLE 80. Mutagenic Cassette: T, G, N CODON Represented I AMINO ACID (Frequency) _______GLYCINE 0 0 0 0 _______ISOLEUCINE0 0 ________PHENYLALANINE 0 TO YES TRYPTOPHAN I PROLINE0 (Frequency) NUN VULAK

(NFL)

0 POLAR2 TGT YES CYSTEINE 2 NONIONIZABLE TOC YES (POL) 0 THREONINE 0 _______ASPARTIC ACID 0 IONIZABLE. ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

0 IONIZABLE: BASIC 0 _______ARGININE. 0 POSITIVE CHARGE HISTIDINE 0 TGA YES STOP CODON I STOP SIGNALI TOTAL 4 2 Amino Acids Are Represented NPL: POL: NEG: POS: STP 1: 2: 0: 0: 1 TABLE 81. Mutagenic Cassette: A, G, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) _______GLYCINE 0 NONPOLAR 0 _______ALANINE 0 -(NFL) 0 ________ISOLEUCINE, 0 0 ________PHENYLALANINE 0- _______TRYPTOPI{AN 0 PROLINE 0 AGT YES SERINE 2 POLAR 2 AGC YES NONIONIZABLE, ________CYSTEINE 0 (POL) ________ASPARAGINE, 0 _______GLUTAMINE 0 _______TYROSINE 0 THREONINE 0 _____ASPARTIC ACID 0 IONIZABLE- ACIDIC 0 GLUJTAMIfC ACID 0 NEGATIVE CHARGE

_(NEG)

0 IONIZABLE. BASIC 2 AGA YES ARGININE, 2 POSITIVE CHARGE AGG YES (POS) HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 TOTAL 4 2 Amino Adids Are Represented NPL: POL: NEG: POS: STP I1 0: 2: 0: 2: 0 135 TABLE 82. Mutagenic Cassette: C/C, C, N CODON Represented AMINO ACID (Frequency) CATEC (Frequcy)

GGC

GGA

GGG

1r.C

YES

YES

YES

uL I U' nuI'lLAR

(NPL)

ALANINE 0 VALINE 0

LEUCINE

ISOLEUCINE

METHIONIN

PHENYLAL

0 E 0 NINE 0

TRYPTO

PROLIN!

SERINE

PHAN 0 0

CYSTEINE

ASPARAGINE 0 _LUTAMENE 0 TYROSINE 0

POLAR

NONIONIZABLE

(POL)

IKIKUNLNt U ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE: BASIC 4 CGT YES ARGININE 4 POSITIVE CHARGE CGC YES (POS) CGA YES COG YES HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 (Si?) TOTAL 8 2 Amino Acids Are Represented NPL: POL: NEG: POS: STP- 4! 0: 0: 4: 0 TABLE 83. Mutagenic Cassette: GiC, C, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 GCT YES ALAIINE 4 (NFL) GCC YES GCA YES GCO YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES CCG YES SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 0 ARGININE o POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 8 2 Amino Acids Are Represented NPL: FOL: NEG: POS: STP 8: 0: 0: 0: 0 136 TABLE 84. Mutagenic Cassette: C/C, A, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 2 CAG YES TYROSINE 0 THREONINE o GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE OAA YES GLUTAMIC ACID 2 (NEG).

GAG YES LYSINE 0 IONIZABLE: BASIC 2 ARGININE O POSITIVE CHARGE CAT YES HISTIDINE 2 (POS) CAC N STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 8 4 Amino Adds Are Represented NPL: POL: NEG: POS: STP 0: 2: 4: 2: 0 TABLE 85. Mutagenic Cassette: GIC, T, N CODON Represented AMINO ACID (Frequency) CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 ALANINE 0 (NPL) GTT YES VALINE 4 GTC YES GTA YES Urn YES CIT YES LEUCINE 4 CYC YES ________ISOLEUCINE 0 TTEMIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GWUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE

(NEG)

LYSINE 0 IONIZABLE. BASIC 0 ARGININE 0 POSITIVE CHARGE -ISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0

(STP)

TOTAL 8 2 Amino Adds Are Represented NPL: POL: NEG: POS: STP 8: 0: 0: 0: 0 137 2.11.2.3. EXONUCLEASE-MEDIATED REASSEMBLY In a particular embodiment, this invention provides for a method for shuffling, assembling, reassembling, recombining, &/or concatenating at least two 00 polynucleotides to form a progeny polynucleotide a chimeric progeny 0 polynucleotide that can be expressed to produce a polypeptide or a gene pathway).

O In a particular embodiment, a double stranded polynucleotide end two single stranded sequences hybridized to each other as hybridization partners) is treated with an exonuclease to liberate nucleotides from one of the two strands, leaving the remaining strand free of its original partner so that, if desired, the remaining strand may be used to achieve hybridization to another partner.

In a particular aspect, a double stranded polynucleotide end (that may be part of- or connected to a polynucleotide or a nonpolynucleotide sequence) is subjected to a source of exonuclease activity. Serviceable sources of exonuclease activity may be an enzyme with 3' exonuclease activity, an enzyme with 5' exonuclease activity, an enzyme with both 3' exonuclease activity and 5' exonuclease activity, and any combination thereof. An exonuclease can be used to liberate nucleotides from one or both ends of a linear double stranded polynucleotide, and from one to all ends of a branched polynucleotide having more than two ends. The mechanism of action of this liberation is believed to be comprised of an enzymatically-catalyzed hydrolysis of terminal nucleotides, and can be allowed to proceed in a time-dependent fashion, allowing experimental control of the progression of the enzymatic process.

By contrast, a non-enzymatic step may be used to shuffle, assemble, reassemble, recombine, and/or concatenate polynucleotide building blocks that is comprised of subjecting a working sample to denaturing (or "melting") conditions (for example, by changing temperature, pH, and /or salinity conditions) so as to melt a working set of double stranded polynucleotides into single polynucleotide strands.

For shuffling, it is desirable that the single polynucleotide strands participate to some extent in annealment with different hybridization partners and not merely revert to exclusive reannealment between what were former partners before the 138 denaturation step). The presence of the former hybridization partners in the reaction vessel, however, does not preclude, and may sometimes even favor, reannealment of a single stranded polynucleotide with its former partner, to recreate an original double stranded polynucleotide.

00 In contrast to this non-enzymatic shuffling step comprised of subjecting

O

c double stranded polynucleotide building blocks to denaturation, followed by CN annealment, the instant invention further provides an exonuclease-based approach 0requiring no denaturation rather, the avoidance of denaturing conditions and the C 10 maintenance of double stranded polynucleotide substrates in annealed nondenatured) state are necessary conditions for the action of exonucleases exonuclease III and red alpha gene product). Additionally in contrast, the generation of single stranded polynucleotide sequences capable of hybridizing to other single stranded polynucleotide sequences is the result of covalent cleavage and hence sequence destruction in one of the hybridization partners. For example, an exonuclease II enzyme may be used to enzymatically liberate 3' terminal nucleotides in one hybridization strand (to achieve covalent hydrolysis in that polynucleotide strand); and this favors hybridization of the remaining single strand to a new partner (since its former partner was subjected to covalent cleavage).

By way of further illustration, a specific exonuclease, namely exonuclease III is provided herein as an example of a 3' exonuclease; however, other exonucleases may also be used, including enzymes with 5' exonuclease activity and enzymes with 3' exonuclease activity, and including enzymes not yet discovered and enzymes not yet developed. It is particularly appreciated that enzymes can be discovered, optimized engineered by directed evolution), or both discovered and optimized specifically for the instantly disclosed approach that have more optimal rates &/or more highly specific activities &/or greater lack of unwanted activities. In fact it is expected that the instant invention may encourage the discovery &/or development of such designer enzymes. In sum, this invention may be practiced with a variety of currently available exonuclease enzymes, as well as enzymes not yet discovered and enzymes not yet developed.

139 The exonuclease action of exonuclease III requires a working double Sstranded polynucleotide end that is either blunt or has a 5' overhang, and the exonuclease action is comprised of enzymatically liberating 3' terminal nucleotides, leaving a single stranded 5' end that becomes longer and longer as the exonuclease 00 action proceeds (see Figure Any 5' overhangs produced by this approach may be Sused to hybridize to another single stranded polynucleotide sequence (which may also be a single stranded polynucleotide or a terminal overhang of a partially double stranded polynucleotide) that shares enough homology to allow hybridization. The 10 ability of these exonuclease III-generated single stranded sequences in overhangs) to hybridize to other single stranded sequences allows two or more polynucleotides to be shuffled, assembled, reassembled, &/or concatenated.

Furthermore, it is appreciated that one can protect the end of a double stranded polynucleotide or render it susceptible to a desired enzymatic action of a serviceable exonuclease as necessary. For example, a double stranded polynucleotide end having a 3' overhang is not susceptible to the exonuclease action ofexonuclease II. However, it may be rendered susceptible to the exonuclease action of exonuclease I by a variety of means; for example, it may be blunted by treatment with a polymerase, cleaved to provide a blunt end or a 5' overhang, joined (ligated or hybridized) to another double stranded polynucleotide to provide a blunt end or a 5' overhang, hybridized to a single stranded polynucleotide to provide a blunt end or a 5' overhang, or modified by any of a variety of means).

According to one aspect, an exonuclease may be allowed to act on one or on both ends of a linear double stranded polynucleotide and proceed to completion, to near completion, or to partial completion. When the exonuclease action is allowed to go to completion, the result will be that the length of each 5' overhang will extend far towards the middle region of the polynucleotide in the direction of what might be considered a "rendezvous point" (which may be somewhere near the polynucleotide midpoint). Ultimately, this results in the production of single stranded polynucleotides (that can become dissociated) that are each about half the length of 140 S* the original double stranded polynucleotide (see Figure Alternatively, an exonuclease-mediated reaction can be terminated before proceeding to completion.

Thus this exonuclease-mediated approach is serviceable for shuffling, assembling &/or reassembling, recombining, and concatenating polynucleotide

O

Sbuilding blocks, which polynucleotide building blocks can be up to ten bases long or c tens of bases long or hundreds of bases long or thousands of bases long or tens of C' thousands of bases long or hundreds of thousands of bases long or millions of bases Slong or even longer.

CN This exonuclease-mediated approach is based on the action of double stranded DNA specific exodeoxyribonuclease activity ofE. coli exonuclease III.

Substrates for exonuclease I may be generated by subjecting a double stranded polynucleotide to fragmentation. Fragmentation may be achieved by mechanical means shearing, sonication, etc.), by enzymatic means using restriction enzymes), and by any combination thereof. Fragments of a larger polynucleotide may also be generated by polymerase-mediated synthesis.

Exonuclease III is a 28K monomeric enzyme, product of the xthA gene of E.

coli with four known activities: exodeoxyribonuclease (alternatively referred to as exonuclease herein), RNaseH, DNA-3'-phosphatase, and AP endonuclease. The exodeoxyribonuclease activity is specific for double stranded DNA. The mechanism of action is thought to involve enzymatic hydrolysis of DNA from a 3' end progressively towards a 5' direction, with formation of nucleoside 5'-phosphates and a residual single strand. The enzyme does not display efficient hydrolysis of single stranded DNA, single-stranded RNA, or double-stranded RNA; however it degrades RNA in an DNA-RNA hybrid releasing nucleoside 5'-phosphates. The enzyme also releases inorganic phosphate specifically from 3'phosphomonoester groups on DNA, but not from RNA or short oligonucleotides. Removal of these groups converts the terminus into a primer for DNA polymerase action.

Additional examples of enzymes with exonuclease activity include red-alpha and venom phosphodiesterases. Red alpha (reda) gene product (also referred to as 141 Slambda exonuclease) is of bacteriophage X origin. The redo gene is transcribed from the leftward promoter and its product is involved (24 kD) in recombination.

Red alpha gene product acts processively from 5'-phosphorylated termini to liberate mononucleotides from duplex DNA (Takahashi Kobayashi, 1990). Venom phosphodiesterases (Laskowski, 1980) is capable of rapidly opening supercoiled

DNA.

CI It is appreciated that related but nonidentical nucleic acid strands can O hybridize as a step towards the generation of chimeric molecules. However, because CI 10 they are non-dentical, they can form what might be termed a heteromeric complex, i.e. an annealment of non-identical nucleic acids. In this complex, it is appreciated that even though two heterologous strands may be hybridized in part, terminal sequences of sufficiently heterologous strands will not hybridize, hence they are hybridizable. This poses a problem, because unhybrizided ends are suboptimal for priming extension and for serving as points ofligation. Accordingly, in another embodiment, this invention provides for the use of exonuclease treatment as a means to liberate 3' and 5'-terminal nucleotides from the unhybridized single-stranded end of an annealed nucleic acid strand in a heteromeric nucleic acid complex, leaving a shortened but hybridized end to facilitate polymerase-based extension and/or ligasemediated ligation of the treated end. This procedure is nicknamed "pruning of loose ends" A variety ofexonucleases are serviceable for this "pruning" purpose. Thus, particularly preferred exonuclease treatments for this "pruning" purpose according to this invention include treatment with Mung Bean Nuclease, treatment with S Nuclease, and treatment with E.coli DNA. Additional preferred exonuclease treatments for this "pruning" purpose according to this invention include the use of the enzymes listed below (selected enzyme properties are provided).

142 Exonucleases Enzyme Name (exemplary commercial source other 5-3- 3 5' 5-3- Strand comments) Exonuclease Exonuclease Polymerase Displacement Km dNT~s VentR DNA Polymerase' No Yes Yes Yes' 60 e Deep Ventp. DNA POlVMerasea No Yes Yes 50 Ue E. coli DNA Polymerase lb Yes Yes Yes No 1-2 uM' Kienow Fragment DNA No Yes Yes Yes 2 IuMs Polymerase Ib' T4 DNA Polymerase' No Yes Yes No 2 jamb T7 DNA Polymerase' No Yes Yes No 18 Um' Taq DNA Polymerase b Yes No Yes No 13 ,uM Mung Bean Nuclease bk No Yes Yes SI Nuclease No Yes Yes Pfu DNA Polyinerase b No Yes Yes Ti DNA Polymeraseb No Yes Yes rBst DNA Polymerasec Yes No Yes Pwo DNA Polymerase d No Yes Yes Exonuclease I No Yes Exonuclease Mcl No Yes 'atagene bpromega 'Epicenter d Roche 'Kong, H. Kucera, R. and Jack, (1993) J. Biol. Chem. 268, 1965-1975.

fMcClure, W R. and Jovin, T. (1975) J. Biol. Chem 250, 4073-4080.

9Polesky A. Steitz, T. Grindley, N. D. F. and Joyce, C. (1990) J. Biol. Chem. 265, 14579-14591.

'Gillin, F. D. and Nossal, N. (1975) Biochem. Biophys. Res. Cominun 64, 457-464.

'Patel, S. Wong, E. and Johnson, IC (1991) Biochemistry 30, 511-525.

k exhibits some double-stranded exonuclease activity from both ends at higher concentrations of enzyme Mexhibits some double-stranded exonuclease activity at higher concentrations of enzyme In another preferred embodiment, this invention provides that nucleic acid N building blocks for exonuclease-mediated reassembly include nucleic acid strands that upon hybridizing to non-identical nucleic acid strands form heteromeric complexes. Within these complexes, a strand that is annealed to more than one other strand is referred to as a poly-binding strand, and a strand 00 that is annealed to only one other strand is referred to as a mono-binding strand.

SAccordingly, mono-binding strands are usually, but not always shorter in length Sthan poly-binding strands. By way of non-liming exemplification, both monobinding strands and poly-binding strands can be generated from a template progenitor molecule by either synthesis, fragmentation (physical or enzymebased), isolation by selective treatment with Dpn I) &/or by denaturation.

144 2.11.2.3. NON-STOCHASTIC LIGATION REASSEMBLY SIn one aspect, the present invention provides a non-stochastic method termed synthetic ligation reassembly (SLR), that is somewhat related to stochastic shuffling, save that the nucleic acid building blocks are not shuffled or concatenated or Schimerized randomly, but rather are assembled non-stochastically.

C A particularly glaring difference is that the instant SLR method does not depend on the presence of a high level of homology between polynucleotides to be C' 10 shuffled. In contrast, prior methods, particularly prior stochastic shuffling methods require that presence of a high level of homology, particularly at coupling sites, between polynucleotides to be shuffled. Accordingly these prior methods favor the regeneration of the original progenitor molecules, and are suboptimal for generating large numbers of novel progeny chimeras, particularly full-length progenies. The instant invention, on the other hand, can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10100 different chimeras.

Conceivably, SLR can even be used to generate libraries comprised of over 101 0 00 different progeny chimeras with (no upper limit in sight).

Thus, in one aspect, the present invention provides a method, which method is non-stochastic, of producing a set of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design, which method is comprised of the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be "serviceable" for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders. Thus, in one aspect, the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends and, if more than one 145 assembly step is to be used, then the overall assembly order in which the nucleic Sacid building blocks can be coupled is also specified by the sequential order of the assembly step(s). Figure 4, Panel C illustrates an exemplary assembly process comprised of 2 sequential steps to achieve a designed (non-stochastic) overall assembly order for five nucleic acid building blocks. In a preferred embodiment of this invention, the annealed building pieces are treated with an enzyme, such as a Sligase T4 DNA ligase), achieve covalent bonding of the building pieces.

SIn a preferred embodiment, the design of nucleic acid building blocks is

C

10 obtained upon analysis of the sequences of a set of progenitor nucleic acid templates that serve as a basis for producing a progeny set of finalized chimeric nucleic acid molecules. These progenitor nucleic acid templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, i.e. chimerized or shuffled.

In one exemplification, this invention provides for the chimerization of a family of related genes and their encoded family of related products. In a particular exemplification, the encoded products are enzymes. As a representative list of families of enzymes which may be mutagenized in accordance with the aspects of the present invention, there may be mentioned, the following enzymes and their functions: 1 Lipase/Esterase a. Enantioselective hydrolysis of esters (lipids)/ thioesters 1) Resolution ofracemic mixtures 2) Synthesis of optically active acids or alcohols from mesodiesters b. Selective syntheses 1) Regiospecific hydrolysis of carbohydrate esters 2) Selective hydrolysis of cyclic secondary alcohols c. Synthesis of optically active esters, lactones, acids, alcohols 1) Transesterification of activated/nonactivated esters 146 2) Interesterification 3) Optically active lactones from hydroxyesters 4) Regio- and enantioselective ring opening of anhydrides d. Detergents e. Fat/Oil conversion 00 f. Cheese ripening C 2 Protease a. Ester/amide synthesis C 10 b. Peptide synthesis c. Resolution of racemic mixtures of amino acid esters d. Synthesis of non-natural amino acids e. Detergents/protein hydrolysis 3 Glycosidase/Glycosyl transferase a. Sugar/polymer synthesis b. Cleavage of glycosidic linkages to form mono, di-and oligosaccharides c. Synthesis of complex oligosaccharides d. Glycoside synthesis using UDP-galactosyl transferase e. Transglycosylationofdisaccharides, glycosyl fluorides, aryl galactosides f. Glycosyl transfer in oligosaccharide synthesis g. Diastereoselective cleavage of P-glucosylsulfoxides h. Asymmetric glycosylations i. Food processing j. Paper processing 4 Phosphatase/Kinase a. Synthesis/hydrolysis of phosphate esters 1) Regio-, enantioselective phosphorylation 2) Introduction of phosphate esters 147 O 3) Synthesize phospholipid precursors 4) Controlled polynucleotide synthesis b. Activate biological molecule c. Selective phosphate bond formation without protecting groups 0 5 Mono/Dioxygenase c a. Direct oxyfunctionalization of unactivated organic substrates b. Hydroxylation of alkane, aromatics, steroids c. Epoxidation of alkenes C 10 d. Enantioselective sulphoxidation e. Regio- and stereoselective Bayer-Villiger oxidations 6 Haloperoxidase a. Oxidative addition of halide ion to nucleophilic sites b. Addition of hypohalous acids to olefinic bonds c. Ring cleavage of cyclopropanes d. Activated aromatic substrates converted to ortho and para derivatives e. 1.3 diketones converted to 2-halo-derivatives f. Heteroatom oxidation of sulfur and nitrogen containing substrates g. Oxidation of enol acetates, alkynes and activated aromatic rings 7 Lignin peroxidase/Diarylpropane peroxidase a. Oxidative cleavage of C-C bonds b. Oxidation ofbenzylic alcohols to aldehydes c. Hydroxylation of benzylic carbons d. Phenol dimerization e. Hydroxylation of double bonds to form diols f. Cleavage of lignin aldehydes 8 Epoxide hydrolase a. Synthesis of enantiomerically pure bioactive compounds b. Regio- and enantioselective hydrolysis of epoxide 148 CI c. Aromatic and olefinic epoxidation by monooxygenases to form epoxides Resolution of racemic epoxides e. Hydrolysis of steroid epoxides 00 9 Nitrile hydratase/nitrilase a. Hydrolysis of aliphatic nitriles to carboxamides C b. Hydrolysis of aromatic, heterocyclic, unsaturated aliphatic nitriles to Scorresponding acids c 10 c. Hydrolysis of acrylonitrile d. Production of aromatic and carboxamides, carboxylic acids (nicotinamide, picolinamide, isonicotinamide) e. Regioselective hydrolysis of acrylic dinitrile f. a-amino acids from a-hydroxynitriles Transaminase a. Transfer of amino groups into oxo-acids 11 Amidase/Acylase a. Hydrolysis of amides, amidines, and other C-N bonds b. Non-natural amino acid resolution and synthesis These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

Thus according to one aspect of this invention, the sequences of a plurality of progenitor nucleic acid templates are aligned in order to select one or more demarcation points, which demarcation points can be located at an area of homology, and are comprised of one or more nucleotides, and which demarcation points are shared by at least two of the progenitor templates. The demarcation points can be used to delineate the boundaries of nucleic acid building blocks to be 149 generated. Thus, the demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the progeny molecules.

Preferably a serviceable demarcation point is an area of homology 00oO (comprised of at least one homologous nucleotide base) shared by at least two Cc progenitor templates. More preferably a serviceable demarcation point is an area of N, homology that is shared by at least half of the progenitor templates. More preferably Sstill a serviceable demarcation point is an area of homology that is shared by at least C 10 two thirds of the progenitor templates. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the progenitor templates. Even more preferably still a serviceable demarcation points is an area of homology that is shared by at almost all of the progenitor templates. Even more preferably still a serviceable demarcation point is an area of homology that is shared by all of the progenitor templates.

The process of designing nucleic acid building blocks and of designing the mutually compatible ligatable ends of the nucleic acid building blocks to be assembled is illustrated in Figures 6 and 7. As shown, the alignment of a set of progenitor templates reveals several naturally occurring demarcation points, and the identification of demarcation points shared by these templates helps to nonstochastically determine the building blocks to be generated and used for the generation of the progeny chimeric molecules.

In a preferred embodiment, this invention provides that the ligation reassembly process is performed exhaustively in order to generate an exhaustive library. In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules. At the same time, in a particularly preferred embodiment, the assembly order the order of assembly of each building block in the 5' to 3' sequence of each finalized chimeric nucleic acid) in each combination is by design (or nonstochastic). Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.

In another preferred embodiment, this invention provides that the ligation reassembly process is performed systematically, for example in order to generate a systematically compartmentalized library, with compartments that can be screened Cc systematically, e.g. one by one. In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with Sthe selective and judicious use of sequentially stepped assembly reactions, an C 10 experimental design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, it allows a potentially very large number of progeny molecules to be examined systematically in smaller groups.

Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, the instant invention provides for the generation of a library (or set) comprised of a large number of progeny molecules.

Because of the non-stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design. In a particularly preferred embodiment of this invention, such a generated library is comprised of preferably greater than 10 3 different progeny molecular species, more preferably greater than 105 different progeny molecular species, more preferably still greater than 1010 different progeny molecular species, more preferably still greater than 1015 different progeny molecular species, more preferably still greater than 1020 different progeny molecular species, more preferably still greater than 1030 different progeny molecular species, more preferably still greater than 1040 different progeny molecular species, more preferably still greater than 1050 different progeny molecular species, more preferably still greater than 1060 different progeny molecular species, more preferably still greater than 1070 different progeny molecular species, more preferably still greater than 1080 different progeny 151 molecular species, more preferably still greater than 10' 0 0 different progeny molecular species, more preferably still greater than 10110 different progeny molecular species, more preferably still greater than 10120 different progeny molecular species, more preferably still greater than 10130 different progeny 00 5 molecular species, more preferably still greater than 10140 different progeny molecular species, more preferably still greater than 10150 different progeny O molecular species, more preferably still greater than 10175 different progeny molecular species, more preferably still greater than 10200 different progeny Smolecular species, more preferably still greater than 10300 different progeny So molecular species, more preferably still greater than 104 00 different progeny molecular species, more preferably still greater than 105 00 different progeny molecular species, and even more preferably still greater than 101000 different progeny molecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules, produced as described is comprised of a polynucleotide encoding a polypeptide. According to one preferred embodiment, this polynucleotide is a gene, which may be a man-made gene. According to another preferred embodiment, this polynucleotide is a gene pathway, which may be a man-made gene pathway. This invention provides that one or more man-made genes generated by this invention may be incorporated into a man-made gene pathway, such as a pathway operable in a eukaryotic organism (including a plant).

It is appreciated that the power of this invention is exceptional, as there is much freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template.

152 Alternatively, a codon can be altered such that the coding for an original amino acid is altered. This invention provides that such substitutions can be introduced into the _nucleic acid building block in order to increase the incidence of intermolecularly homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of 0O progeny chimeric molecules to be generated.

O

N In another exemplifaction, the synthetic nature of the step in which the Sbuilding blocks are generated allows the design and introduction ofnucleotides (e.g.

C N 10 one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process by mutageneis) or in an in vivo process by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.

Thus, according to another embodiment, this invention provides that a nucleic acid building block can be used to introduce an intron. Thus, this invention provides that functional introns may be introduced into a man-made gene of this invention. This invention also provides that functional introns may be introduced into a man-made gene pathway of this invention. Accordingly, this invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially introduced intron(s).

Accordingly, this invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced intron(s). Preferably, the artificially introduced intron(s) are functional in one or more host cells for gene splicing much in the way that naturallyoccurring introns serve functionally in gene splicing. This invention provides a process of producing man-made intron-containing polynucleotides to be introduced into host organisms for recombination and/or splicing.

153 SThe ability to achieve chimerizations, using couplings as described herein, in areas of little or no homology among the progenitor molecules, is particularly useful, and in fact critical, for the assembly of novel gene pathways. This invention thus provides forthe generation of novel man-made gene pathways using synthetic ligation reassembly. In a particular aspect, this is achieved by the introduction of 00 regulatory sequences, such as promoters, that are operable in an intended host, to Sconfer operability to a novel gene pathway when it is introduced into the intended N host. In a particular exemplification, this invention provides for the generation of Snovel man-made gene pathways that is operable in a plurality of intended hosts (e.g.

N 10 in a microbial organism as well as in a plant cell). This can be achieved, for example, by the introduction of a plurality of regulatory sequences, comprised of a regulatory sequence that is operable in a first intended host and a regulatory sequence that is operable in a second intended host. A similar process can be performed to achieve operability of a gene pathway in a third intended host species, etc. The number of intended host species can be each integer from 1 to 10 or alternatively over 10. Alternatively, for example, operability of a gene pathway in a plurality of intended hosts can be achieved by the introduction of a regulatory sequence having intrinsic operability in a plurality of intended hosts.

Thus, according to a particular embodiment, this invention provides that a nucleic acid building block can be used to introduce a regulatory sequence, particularly a regulatory sequence for gene expression. Preferred regulatory sequences include, but are not limited to, those that are man-made, and those found in archeal, bacterial, eukaryotic (including mitochondrial), viral, and prionic or prion-like organisms. Preferred regulatory sequences include but are not limited to, promoters, operators, and activator binding sites. Thus, this invention provides that functional regulatory sequences may be introduced into a man-made gene of this invention. This invention also provides that functional regulatory sequences may be introduced into a man-made gene pathway of this invention.

Accordingly, this invention provides for the generation of a chimeric polynucleotide that is a man-made gene containing one (or more) artificially 154 O introduced regulatory sequence(s). Accordingly, this invention also provides for the generation of a chimeric polynucleotide that is a man-made gene pathway containing one (or more) artificially introduced regulatory sequence(s). Preferably, an artificially introduced regulatory sequence(s) is operatively linked to one or more genes in the man-made polynucleotide, and are functional in one or more host cells.

00 c Preferred bacterial promoters that are serviceable for this invention include c, lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Serviceable eukaryotic promoters 0 include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs 10 from retrovirus, and mouse metallothionein-I. Particular plant regulatory sequences include promoters active in directing transcription in plants, either constitutively or stage and/or tissue specific, depending on the use of the plant or parts thereof. These promoters include, but are not limited to promoters showing constitutive expression, such as the 35S promoter of Cauliflower Mosaic Virus (CaMV) (Guilley et al., 1982), those for leaf-specific expression, such as the promoter of the ribulose bisphosphate carboxylase small subunit gene (Coruzzi et al., 1984), those for rootspecific expression, such as the promoter from the glutamin synthase gene (Tingey et al., 1987), those for seed-specific expression, such as the cruciferin A promoter from Brassica napus (Ryan et al., 1989), those for tuber-specific expression, such as the class-I patatin promoter from potato (Rocha-Sasa et al., 1989; Wenzler et al., 1989) or those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato (Bird et al., 1988).

Other regulatory sequences that are preferred for this invention include terminator sequences and polyadenylation signals and any such sequence functioning as such in plants, the choice of which is within the level of the skilled artisan. An example of such sequences is the 3' flanking region of the nopaline synthase (nos) gene ofAgrobacterium tumefaciens (Bevan, 1984). The regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV, and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Cirus (AIMV) RNA4 (Brederode et al., 1980) or any other sequences functioning in a like manner.

155 Man-made genes produced using this invention can also serve as a substrate for recombination with another nucleic acid. Likewise, a man-made gene pathway produced using this invention can also serve as a substrate for recombination with another nucleic acid. In a preferred instance, the recombination is facilitated by, or 00 occurs at, areas of homology between the man-made intron-containing gene and a Cc nucleic acid with serves as a recombination partner. In a particularly preferred instance, the recombination partner may also be a nucleic acid generated by this Sinvention, including a man-made gene or a man-made gene pathway.

N, 10 Recombination may be facilitated by or may occur at areas of homology that exist at the one (or more) artificially introduced intron(s) in the man-made gene.

The synthetic ligation reassembly method of this invention utilizes a plurality of nucleic acid building blocks, each of which preferably has two ligatable ends.

The two ligatable ends on each nucleic acid building block may be two blunt ends each having an overhang of zero nucleotides), or preferably one blunt end and one overhang, or more preferably still two overhangs.

A serviceable overhang for this purpose may be a 3' overhang or a overhang. Thus, a nucleic acid building block may have a 3' overhang or alternatively a 5' overhang or alternatively two 3' overhangs or alternatively two overhangs. The overall order in which the nucleic acid building blocks are assembled to form a finalized chimeric nucleic acid molecule is determined by purposeful experimental design and is not random.

According to one preferred embodiment, a nucleic acid building block is generated by chemical synthesis of two single-stranded nucleic acids (also referred to as single-stranded oligos) and contacting them so as to allow them to anneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size. The sizes of these building blocks can be small or large depending on the choice of the 156 Sexperimenter. Preferred sizes for building block range from 1 base pair (not including any overhangs) to 100,000 base pairs (not including any overhangs).

Other preferred size ranges are also provided, which have lower limits of from 1 bp to 10,000 bp (including every integer value in between), and upper limits of from 2 bp to 100, 000 bp (including every integer value in between).

00 M It is appreciated that current methods ofpolymerase-based amplification can CN be used to generate double-stranded nucleic acids of up to thousands of base pairs, if not tens of thousands of base pairs, in length with high fidelity. Chemical synthesis CN 10 phosphoramidite-based) can be used to generate nucleic acids of up to hundreds ofnucleotides in length with high fidelity; however, these can be assembled, e.g.

using overhangs or sticky ends, to form double-stranded nucleic acids of up to thousands of base pairs, if not tens of thousands of base pairs, in length if so desired.

A combination of methods phosphoramidite-based chemical synthesis and PCR) can also be used according to this invention. Thus, nucleic acid building block made by different methods can also be used in combination to generate a progeny molecule of this invention.

The use of chemical synthesis to generate nucleic acid building blocks is particularly preferred in this invention is advantageous for other reasons as well, including procedural safety and ease. No cloning or harvesting or actual handling of any biological samples is required. The design of the nucleic acid building blocks can be accomplished on paper. Accordingly, this invention teaches an advance in procedural safety in recombinant technologies.

Nonetheless, according to one preferred embodiment, a double-stranded nucleic acid building block according to this invention may also be generated by polymerase-based amplification of a polynucleotide template. In a non-limiting exemplification, as illustrated in Figure 2, a first polymerase-based amplification reaction using a first set of primers, F 2 and Ri, is used to generate a blunt-ended product (labeled Reaction 1, Product which is essentially identical to Product A.

157 (A second polymerase-based amplification reaction using a second set of primers, Fi and R 2 is used to generate a blunt-ended product (labeled Reaction 2, Product 2), Swhich is essentially identical to Product B. These two products are mixed and allowed to melt and anneal, generating potentially useful double-stranded nucleic acid building blocks with two overhangs. In the example of Fig. 2, the product with 00 the 3' overhangs (Product C) is selected by nuclease-based degradation of the other 3 products using a 3' acting exonuclease, such as exonuclease III. It is appreciated I that a 5' acting exonuclease red alpha) may be also be used, for example to Sselect Product D instead. It is also appreciated that other selection means can also N 10 be used, including hybridization-based means, and that these means can incorporate a further means, such as a magnetic bead-based means, to facilitate separation of the desired product.

Many other methods exist by which a double-stranded nucleic acid building block can be generated that is serviceable for this invention; and these are known in the art and can be readily performed by the skilled artisan.

According to particularly preferred embodiment, a double-stranded nucleic acid building block that is serviceable for this invention is generated by first generating two single stranded nucleic acids and allowing them to anneal to form a double-stranded nucleic acid building block. The two strands of a double-stranded nucleic acid building block may be complementary at every nucleotide apart from any that form an overhang; thus containing no mismatches, apart from any overhang(s). According to another embodiment, the two strands of a doublestranded nucleic acid building block are complementary at fewer than every nucleotide apart from any that form an overhang. Thus, according to this embodiment, a double-stranded nucleic acid building block can be used to introduce codon degeneracy. Preferably the codon degeneracy is introduced using the sitesaturation mutagenesis described herein, using one or more N,N,G/T cassettes or alternatively using one or more N,N,N cassettes.

158 SContained within an exemplary experimental design for achieving an ordered Sassembly according to this invention are: 1) The design of specific nucleic acid building blocks.

2) The design of specific ligatable ends on each nucleic acid building block.

00 3) The design of a particular order of assembly of the nucleic acid building Sblocks.

SAn overhang may be a 3' overhang or a 5' overhang. An overhang may also Ci 10 have a terminal phosphate group or alternatively may be devoid of a terminal phosphate group (having, a hydroxyl group instead). An overhang may be comprised of any number of nucleotides. Preferably an overhang is comprised of 0 nucleotides (as in a blunt end) to 10,000 nucleotides. Thus, a wide range of overhang sizes may be serviceable. Accordingly, the lower limit may be each integer from 1-200 and the upper limit may be each integer from 2-10,000.

According to a particular exemplification, an overhang may consist of anywhere from I nucleotide to 200 nucleotides (including every integer value in between).

The final chimeric nucleic acid molecule may be generated by sequentially assembling 2 or more building blocks at a time until all the designated building blocks have been assembled. A working sample may optionally be subjected to a process for size selection or purification or other selection or enrichment process between the performance of two assembly steps. Alternatively, the final chimeric nucleic acid molecule may be generated by assembling all the designated building blocks at once in one step.

In vitro.Shufljng The equivalents of some standard genetic matings may also be performed by shuffling in vitro. For example, a "molecular backcross" can be performed by repeatedly mixing the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest. As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice.

159 SIt is useful, for example, for the removal of neutral mutations that affect unselected characteristics immunogenicity). Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not, an advantage which cannot be achieved by error-prone mutagenesis or cassette mutagenesis methods.

00 CM Large, functional genes can be assembled correctly from a mixture of small random polynucleotides. This reaction may be of use for the reassembly of genes Sfrom the highly fragmented DNA of fossils. In addition random nucleic acid c 10 fragments from fossils may be combined with polynucleotides from similar genes from related species.

It is also contemplated that the method of this invention can be used for the in vitro amplification of a whole genome from a single cell as is needed for a variety of research and diagnostic applications. DNA amplification by PCR is in practice limited to a length of about 40 kb. Amplification of a whole genome such as that of E. coli 000 kb) by PCR would require about 250 primers yielding 125 forty kb polynucleotides. This approach is not practical due to the unavailability of sufficient sequence data. On the other hand, random production of polynucleotides of the genome with sexual PCR cycles, followed by gel purification of small polynucleotides will provide a multitude of possible primers. Use of this mix of random small polynucleotides as primers in a PCR reaction alone or with the whole genome as the template should result in an inverse chain reaction with the theoretical endpoint of a single concatemer containing many copies of the genome.

100 fold amplification in the copy number and an average polynucleotide size of greater than 50 kb may be obtained when only random polynucleotides are used. It is thought that the larger concatemer is generated by overlap of many smaller polynucleotides. The quality of specific PCR products obtained using synthetic primers will be indistinguishable from the product obtained from unamplified DNA. It is expected that this approach will be useful for the mapping of genomes.

160 SThe polynucleotide to be shuffled can be produced as random or non-random Spolynucleotides, at the discretion of the practitioner.

00 In vivoShufflling c In an embodiment of in vivo shuffling, the mixed population of the specific N nucleic acid sequence is introduced into bacterial or eukaryotic cells under 0conditions such that at least two different nucleic acid sequences are present in each host cell. The polynucleotides can be introduced into the host cells by a variety of different methods. The host cells can be transformed with the smaller polynucleotides using methods known in the art, for example treatment with calcium chloride. If the polynucleotides are inserted into a phage genome, the host cell can be transfected with the recombinant phage genome having the specific nucleic acid sequences. Alternatively, the nucleic acid sequences can be introduced into the host cell using electroporation, transfection, lipofection, biolistics, conjugation, and the like.

In general, in this embodiment, the specific nucleic acids sequences will be present in vectors which are capable of stably replicating the sequence in the host cell. In addition, it is contemplated that the vectors will encode a marker gene such that host cells having the vector can be selected. This ensures that the mutated specific nucleic acid sequence can be recovered after introduction into the host cell.

However, it is contemplated that the entire mixed population of the specific nucleic acid sequences need not be present on a vector sequence. Rather only a sufficient number of sequences need be cloned into vectors to ensure that after introduction of the polynucleotides into the host cells each host cell contains one vector having at least one specific nucleic acid sequence present therein. It is also contemplated that rather than having a subset of the population of the specific nucleic acids sequences cloned into vectors, this subset may be already stably integrated into the host cell.

161 SIt has been found that when two polynucleotides which have regions of identity are inserted into the host cells homologous recombination occurs between the two polynucleotides. Such recombination between the two mutated specific nucleic acid sequences will result in the production of double or triple hybrids in some situations.

00 M It has also been found that the frequency of recombination is increased if N, some of the mutated specific nucleic acid sequences are present on linear nucleic Sacid molecules. Therefore, in a preferred embodiment, some of the specific nucleic C 10 acid sequences are present on linear polynucleotides.

After transformation, the host cell transformants are placed under selection to identify those host cell transformants which contain mutated specific nucleic acid sequences having the qualities desired. For example, if increased resistance to a particular drug is desired then the transformed host cells may be subjected to increased concentrations of the particular drug and those transformants producing mutated proteins able to confer increased drug resistance will be selected. If the enhanced ability of a particular protein to bind to a receptor is desired, then expression of the protein can be induced from the transformants and the resulting protein assayed in a ligand binding assay by methods known in the art to identify that subset of the mutated population which shows enhanced binding to the ligand.

Alternatively, the protein can be expressed in another system to ensure proper processing.

Once a subset of the first recombined specific nucleic acid sequences (daughter sequences) having the desired characteristics are identified, they are then subject to a second round of recombination.

In the second cycle of recombination, the recombined specific nucleic acid sequences may be mixed with the original mutated specific nucleic acid sequences (parent sequences) and the cycle repeated as described above. In this way a set of second recombined specific nucleic acids sequences can be identified which have 162 Q enhanced characteristics or encode for proteins having enhanced properties. This cycle can be repeated a number of times as desired.

It is also contemplated that in the second or subsequent recombination cycle, a backcross can be performed. A molecular backcross can be performed by mixing the desired specific nucleic acid sequences with a large number of the wild-type Csequence, such that at least one wild-type nucleic acid sequence and a mutated

O

N nucleic acid sequence are present in the same host cell after transformation.

O Recombination with the wild-type specific nucleic acid sequence will eliminate

O

N 10 those neutral mutations that may affect unselected characteristics such as immunogenicity but not the selected characteristics.

In another embodiment of this invention, it is contemplated that during the first round a subset of the specific nucleic acid sequences can be generated as smaller polynucleotides by slowing or halting their PCR amplification prior to introduction into the host cell. The size of the polynucleotides must be large enough to contain some regions of identity with the other sequences so as to homologously recombine with the other sequences. The size of the polynucleotides will range from 0.03 kb to 100 kb more preferably from 0. 2 kb to 10 kb. It is also contemplated that in subsequent rounds, all of the specific nucleic acid sequences other than the sequences selected from the previous round may be utilized to generate PCR polynucleotides prior to introduction into the host cells.

The shorter polynucleotide sequences can be single-stranded or double-stranded. If the sequences were originally single-stranded and have become double-stranded they can be denatured with heat, chemicals or enzymes prior to insertion into the host cell. The reaction conditions suitable for separating the strands of nucleic acid are well known in the art.

The steps of this process can be repeated indefinitely, being limited only by the number of possible hybrids which can be achieved. After a certain number of 163 cycles, all possible hybrids will have been achieved and further cycles are redundant.

In an embodiment the same mutated template nucleic acid is repeatedly recombined and the resulting recombinants selected for the desired characteristic.

00 Therefore, the initial pool or population of mutated template nucleic acid is

C

c cloned into a vector capable of replicating in a bacteria such as E. coli. The I particular vector is not essential, so long as it is capable of autonomous replication in SE. coli. In a preferred embodiment, the vector is designed to allow the expression 10 and production of any protein encoded by the mutated specific nucleic acid linked to the vector. It is also preferred that the vector contain a gene encoding for a selectable marker.

The population of vectors containing the pool of mutated nucleic acid sequences is introduced into the E. coli host cells. The vector nucleic acid sequences may be introduced by transformation, transfection or infection in the case of phage. The concentration of vectors used to transform the bacteria is such that a number of vectors is introduced into each cell. Once present in the cell, the efficiency of homologous recombination is such that homologous recombination occurs between the various vectors. This results in the generation of hybrids (daughters) having a combination of mutations which differ from the original parent mutated sequences.

The host cells are then clonally replicated and selected for the marker gene present on the vector. Only those cells having a plasmid will grow under the selection.

The host cells which contain a vector are then tested for the presence of favorable mutations. Such testing may consist of placing the cells under selective pressure, for example, if the gene to be selected is an improved drug resistance gene.

If the vector allows expression of the protein encoded by the mutated nucleic acid sequence, then such selection may include allowing expression of the protein so 164 encoded, isolation of the protein and testing of the protein to determine whether, for example, it binds with increased efficiency to the ligand of interest.

Once a particular daughter mutated nucleic acid sequence has been identified which confers the desired characteristics, the nucleic acid is isolated either already 00 linked to the vector or separated from the vector. This nucleic acid is then mixed Swith the first or parent population of nucleic acids and the cycle is repeated.

It has been shown that by this method nucleic acid sequences having N 10 enhanced desired properties can be selected.

In an alternate embodiment, the first generation of hybrids are retained in the cells and the parental mutated sequences are added again to the cells. Accordingly, the first cycle of Embodiment I is conducted as described above. However, after the daughter nucleic acid sequences are identified, the host cells containing these sequences are retained.

The parent mutated specific nucleic acid population, either as polynucleotides or cloned into the same vector is introduced into the host cells already containing the daughter nucleic acids. Recombination is allowed to occur in the cells and the next generation of recombinants, or granddaughters are selected by the methods described above.

This cycle can be repeated a number of times until the nucleic acid or peptide having the desired characteristics is obtained. It is contemplated that in subsequent cycles, the population of mutated sequences which are added to the preferred hybrids may come from the parental hybrids or any subsequent generation.

In an alternative embodiment, the invention provides a method of conducting a "molecular" backcross of the obtained recombinant specific nucleic acid in order to eliminate any neutral mutations. Neutral mutations are those mutations which do not confer onto the nucleic acid or peptide the desired properties. Such mutations may however confer on the nucleic acid or peptide undesirable characteristics.

165 SAccordingly, it is desirable to eliminate such neutral mutations. The method of this invention provide a means of doing so.

In this embodiment, after the hybrid nucleic acid, having the desired 5 characteristics, is obtained by the methods of the embodiments, the nucleic acid, the oO vector having the nucleic acid or the host cell containing the vector and nucleic acid Cc is isolated.

SThe nucleic acid or vector is then introduced into the host cell with a large 10 excess of the wild-type nucleic acid. The nucleic acid of the hybrid and the nucleic acid of the wild-type sequence are allowed to recombine. The resulting recombinants are placed under the same selection as the hybrid nucleic acid. Only those recombinants which retained the desired characteristics will be selected. Any silent mutations which do not provide the desired characteristics will be lost through recombination with the wild-type DNA. This cycle can be repeated a number of times until all of the silent mutations are eliminated.

Thus the methods of this invention can be used in a molecular backcross to eliminate unnecessary or silent mutations.

The in vivo recombination method of this invention can be performed blindly on a pool of unknown hybrids or alleles of a specific polynucleotide or sequence.

However, it is not necessary to know the actual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genes can be useful for the generation of any useful proteins, for example, interleukin I, antibodies, tPA and growth hormone. This approach may be used to generate proteins having altered specificity or activity. The approach may also be useful for the generation of hybrid nucleic acid sequences, for example, promoter regions, introns, exons, enhancer sequences, 31 untranslated regions or 51 untranslated regions of genes. Thus this approach may be used to generate genes having 166 increased rates of expression. This approach may also be useful in the study of repetitive DNA sequences. Finally, this approach may be useful to mutate ribozymes or aptamers.

Scaffold-like regions separating regions of diversity in proteins may be particularly suitable for the methods of this invention. The conserved scaffold Sdetermines the overall folding by self-association, while displaying relatively Sunrestricted loops that mediate the specific binding. Examples of such scaffolds are Sthe immunoglobulin beta barrel, and the four-helix bundle. The methods of this

C

10 invention can be used to create scaffold-like proteins with various combinations of mutated sequences for binding.

The equivalents of some standard genetic matings may also be performed by the methods of this invention. For example, a "molecular" backcross can be performed by repeated mixing of the hybrid's nucleic acid with the wild-type nucleic acid while selecting for the mutations of interest. As in traditional breeding, this approach can be used to combine phenotypes from different sources into a background of choice. It is useful, for example, for the removal of neutral mutations that affect unselected characteristics immunogenicity). Thus it can be useful to determine which mutations in a protein are involved in the enhanced biological activity and which are not.

Peptide Display Methods The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by peptide display methods, wherein an associated polynucleotide encodes a displayed peptide which is screened for a phenotype for affinity for a predetermined receptor (ligand).

An increasingly important aspect of bio-pharmaceutical drug development and molecular biology is the identification of peptide structures, including the 167 primary amino acid sequences, ofpeptides or peptidomimetics that interact with biological macromolecules. one method of identifying peptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule a receptor), involves the screening of a large library or peptides for individual library members which possess the desired structure or 00 functional property conferred by the amino acid sequence of the peptide.

Ci In addition to direct chemical synthesis methods for generating peptide Slibraries, several recombinant DNA methods also have been reported. One type C 10 involves the display of a peptide sequence, antibody, or other protein on the surface of a bacteriophage particle or cell. Generally, in these methods each bacteriophage particle or cell serves as an individual library member displaying a single species of displayed peptide in addition to the natural bacteriophage or cell protein sequences.

Each bacteriophage or cell contains the nucleotide sequence information encoding the particular displayed peptide sequence; thus, the displayed peptide sequence can be ascertained by nucleotide sequence determination of an isolated library member.

A well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein. The bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule. The bacteriophage particles library members) which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage sub-population for a subsequent round of affinity enrichment and phage replication. After several rounds of affinity enrichment and phage replication, the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined 168 macromolecule receptor). Such methods are further described in PCT patent publications WO 91/17271, WO 91/18980, WO 91/19818 and WO 93/08278.

The latter PCT publication describes a recombinant DNA method for the display of peptide ligands that involves the production of a library of fusion proteins 00 with each fusion protein composed of a first polypeptide portion, typically ccomprising a variable sequence, that is available for potential binding to a Spredetermined macromolecule, and a second polypeptide portion that binds to DNA, such as the DNA vector encoding the individual fusion protein. When transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the DNA vector encoding it. Upon lysis of the host cell, the fusion protein/vector DNA complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).

Other systems for generating libraries of peptides and like polymers have aspects of both the recombinant and in vitro chemical synthesis methods. In these hybrid methods, cell-free enzymatic machinery is employed to accomplish the in vitro synthesis of the library members peptides or polynucleotides). In one type of method, RNA molecules with the ability to bind a predetermined protein or a predetermined dye molecule were selected by alternate rounds of selection and PCR amplification (Tuerk and Gold, 1990; Ellington and Szostak, 1990). A similar technique was used to identify DNA sequences which bind a predetermined human transcription factor (Thiesen and Bach, 1990; Beaudry and Joyce, 1992; PCT patent publications WO 92/05258 and WO 92/14843). In a similar fashion, the technique of in vitro translation has been used to synthesize proteins of interest and has been proposed as a method for generating large libraries ofpeptides. These methods which rely upon in vitro translation, generally comprising stabilized polysome complexes, are described further in PCT patent publications WO 88/08453, WO 90/05785, WO 90/07003, WO 91/02076, WO 91/05058, and WO 169 92/02536. Applicants have described methods in which library members comprise a fusion protein having a first polypeptide portion with DNA binding activity and a second polypeptide portion having the library member unique peptide sequence; such methods are suitable for use in cell-free in vitro selection formats, among others.

00 C The displayed peptide sequences can be of varying lengths, typically from C, 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and Soften from about 8-15 amino acids long. A library can comprise library members C, 10 having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence. Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kemal, fixed, or the like. The present display methods include methods for in vitro and in vivo display of single-chain antibodies, such as nascent scFv on polysomes or scfv displayed on phage, which enable large-scale screening of scfv libraries having broad diversity of variable region sequences and binding specificities.

The present invention also provides random, pseudorandom, and defined sequence framework peptide libraries and methods for generating and screening those libraries to identify useful compounds peptides, including single-chain antibodies) that bind to receptor molecules or epitopes of interest or gene products that modify peptides or RNA in a desired fashion. The random, pseudorandom, and defined sequence framework peptides are produced from libraries of peptide library members that comprise displayed peptides or displayed single-chain antibodies attached to a polynucleotide template from which the displayed peptide was synthesized. The mode of attachment may vary according to the specific embodiment of the invention selected, and can include encapsulation in a phage particle or incorporation in a cell.

A method of affinity enrichment allows a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected. The polynucleotide can 170 then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just VHI, VLI or CDR portions thereof). Using these methods, one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of shuffling to converge rapidly to a 00 desired high-affinity peptide or scfv. The peptide or antibody can then be g synthesized in bulk by conventional means for any suitable use as a therapeutic C, or diagnostic agent).

cN 10 A significant advantage of the present invention is that no prior information regarding an expected ligand structure is required to isolate peptide ligands or antibodies of interest. The peptide identified can have biological activity, which is meant to include at least specific binding affinity for a selected receptor molecule and, in some instances, will further include the ability to block the binding of other compounds, to stimulate or inhibit metabolic pathways, to act as a signal or messenger, to stimulate or inhibit cellular activity, and the like.

The present invention also provides a method for shuffling a pool of polynucleotide sequences selected by affinity screening a library ofpolysomes displaying nascent peptides (including single-chain antibodies) for library members which bind to a predetermined receptor a mammalian proteinaceous receptor such as, for example, a peptidergic hormone receptor, a cell surface receptor, an intracellular protein which binds to other protein(s) to form intracellular protein complexes such as hetero-dimers and the like) or epitope an immobilized protein, glycoprotein, oligosaccharide, and the like).

Polynucleotide sequences selected in a first selection round (typically by affinity selection for binding to a receptor a ligand)) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences. The recombined selected polynucleotide sequences are subjected to at least one subsequent selection round. The polynucleotide sequences 171 Sselected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to one or more additional rounds of shuffling and subsequent selection.

Selected sequences can also be back-crossed with polynucleotide sequences encoding neutral sequences having insubstantial functional effect on binding), 00 5 such as for example by back-crossing with a wild-type or naturally-occurring sequence substantially identical to a selected sequence to produce native-like functional peptides, which may be less immunogenic. Generally, during back- Scrossing subsequent selection is applied to retain the property of binding to the predetermined receptor (ligand).

Prior to or concomitant with the shuffling of selected sequences, the sequences can be mutagenized. In one embodiment, selected library members are cloned in a prokaryotic vector plasmid, phagemid, or bacteriophage) wherein a collection of individual colonies (or plaques) representing discrete library members are produced. Individual selected library members can then be manipulated by site-directed mutagenesis, cassette mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like) to generate a collection of library members representing a kernal of sequence diversity based on the sequence of the selected library member.

The sequence of an individual selected library member or pool can be manipulated to incorporate random mutation, pseudorandom mutation, defined kernal mutation comprising variant and invariant residue positions and/or comprising variant residue positions which can comprise a residue selected from a defined subset of amino acid residues), codon-based mutation, and the like, either segmentally or over the entire length of the individual selected library member sequence. The mutagenized selected library members are then shuffled by in vitro and/or in vivo recombinatorial shuffling as disclosed herein.

The invention also provides peptide libraries comprising a plurality of individual library members of the invention, wherein each individual library member of said plurality comprises a sequence produced by shuffling of a pool of selected sequences, and each individual library member comprises a variable peptide segment sequence or single-chain antibody segment sequence which is 172 C distinct from the variable peptide segment sequences or single-chain antibody Ssequences of other individual library members in said plurality (although some library members may be present in more than one copy per library due to uneven amplification, stochastic probability, or the like).

00 0 The invention also provides a product-by-process, wherein selected O polynucleotide sequences having (or encoding a peptide having) a predetermined n binding specificity are formed by the process of: screening a displayed peptide or O displayed single-chain antibody library against a predetermined receptor ligand) or epitope antigen macromolecule) and identifying and/or enriching library members which bind to the predetermined receptor or epitope to produce a pool of selected library members, shuffling by recombination the selected library members (or amplified or cloned copies thereof) which binds the predetermined epitope and has been thereby isolated and/or enriched from the library to generate a shuffled library, and screening the shuffled library against the predetermined receptor ligand) or epitope antigen macromolecule) and identifying and/or enriching shuffled library members which bind to the predetermined receptor or epitope to produce a pool of selected shuffled library members.

Antibody Display and Screening Methods The present method can be used to shuffle, by in vitro and/or in vivo recombination by any of the disclosed methods, and in any combination, polynucleotide sequences selected by antibody display methods, wherein an associated polynucleotide encodes a displayed antibody which is screened for a phenotype for affinity for binding a predetermined antigen (ligand).

Various molecular genetic approaches have been devised to capture the vast immunological repertoire represented by the extremely large number of distinct variable regions which can be present in immunoglobulin chains. The naturally-occurring germ line immunoglobulin heavy chain locus is composed of separate tandem arrays of variable segment genes located upstream of a tandem 173 c, array of diversity segment genes, which are themselves located upstream of a tandem array of joining region genes, which are located upstream of the constant region genes. During B lymphocyte development, V-D-J rearrangement occurs wherein a heavy chain variable region gene (VH) is formed by rearrangement to oO 5 form a fused D segment followed by rearrangement with a V segment to form a V-D-J joined product gene which, if productively rearranged, encodes a functional variable region (VH) of a heavy chain. Similarly, light chain loci rearrange one of Sseveral V segments with one of several J segments to form a gene encoding the variable region (VL) of a light chain.

The vast repertoire of variable regions possible in immunoglobulins derives in part from the numerous combinatorial possibilities of joining V and i segments (and, in the case of heavy chain loci, D segments) during rearrangement in B cell development. Additional sequence diversity in the heavy chain variable regions arises from non-uniform rearrangements of the D segments during V-D-J joining and from N region addition. Further, antigen-selection of specific B cell clones selects for higher affinity variants having non-germline mutations in one or both of the heavy and light chain variable regions; a phenomenon referred to as "affinity maturation" or "affinity sharpening". Typically, these "affinity sharpening" mutations cluster in specific areas of the variable region, most commonly in the complementarity-determining regions (CDRs).

In order to overcome many of the limitations in producing and identifying high-affinity immunoglobulins through antigen-stimulated 1 cell development immunization), various prokaryotic expression systems have been developed that can be manipulated to produce combinatorial antibody libraries which may be screened for high-affinity antibodies to specific antigens. Recent advances in the expression of antibodies in Escherichia coli and bacteriophage systems (see "alternative peptide display methods", infra) have raised the possibility that virtually any specificity can be obtained by either cloning antibody genes from characterized hybridomas or by de novo selection using antibody gene libraries from Ig cDNA).

174 SCombinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Huse et al, 1989); Caton and Koprowski, 1990; Mullinax et O0 5 al, 1990; Persson et al, 1991). Various embodiments of bacteriophage antibody display libraries and lambda phage expression libraries have been described (Kang T7 et al, 1991; Clackson et al, 1991; McCafferty et al, 1990; Burton et al, 1991; Hoogenboom etal, 1991; Chang et al, 1991; Breitling et al, 1991; Marks et al, 1991, p. 581; Barbas et al, 1992; Hawkins and Winter, 1992; Marks et al, 1992, p.

779; Marks et al, 1992, p. 16007; and Lowman et al, 1991; Lerner et al, 1992; all incorporated herein by reference). Typically, a bacteriophage antibody display library is screened with a receptor polypeptide, carbohydrate, glycoprotein, nucleic acid) that is immobilized by covalent linkage to a chromatography resin to enrich for reactive phage by affinity chromatography) and/or labeled to screen plaque or colony lifts).

One particularly advantageous approach has been the use of so-called single-chain fragment variable (scfv) libraries (Marks et al, 1992, p. 779; Winter and Milstein, 1991; Clackson et al, 1991; Marks et al, 1991, p. 581; Chaudhary et al, 1990; Chiswell et al, 1992; McCafferty et al, 1990; and Huston et al, 1988).

Various embodiments of scfv libraries displayed on bacteriophage coat proteins have been described.

Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods. The first step generally involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis.

The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s). The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either 175 VH-linker-VL or VL-linker-VH' In principle, the scfv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site.

Thus, scfv fragments are comprised of VH and VL domains linked into a single polypeptide chain by a flexible linker peptide. After the scfv genes are 00oO assembled, they are cloned into a phagemid and expressed at the tip of the M13 r phage (or similar filamentous bacteriophage) as fusion proteins with the N bacteriophage PII (gene 3) coat protein. Enriching for phage expressing an O antibody of interest is accomplished by panning the recombinant phage displaying a C 10 population scfv for binding to a predetermined epitope target antigen, receptor).

The linked polynucleotide of a library member provides the basis for replication of the library member after a screening or selection procedure, and also provides the basis for the determination, by nucleotide sequencing, of the identity of the displayed peptide sequence or VH and VL amino acid sequence. The displayed peptide or single-chain antibody scfv) and/or its VH and VL domains or their CDRs can be cloned and expressed in a suitable expression system. Often polynucleotides encoding the isolated VH and VL domains will be ligated to polynucleotides encoding constant regions (CH and CL) to form polynucleotides encoding complete antibodies chimeric or fully-human), antibody fragments, and the like. Often polynucleotides encoding the isolated CDRs will be grafted into polynucleotides encoding a suitable variable region framework (and optionally constant regions) to form polynucleotides encoding complete antibodies humanized or fully-human), antibody fragments, and the like. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography. Various other uses of such antibodies are to diagnose and/or stage disease neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like.

Various methods have been reported for increasing the combinatorial diversity of a scfv library to broaden the repertoire of binding species (idiotype spectrum) The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunized cells, affording combinatorial diversity in the assortment of VH and VL cassettes 00oO which can be combined. Furthermore, the VH and VL cassettes can themselves be

O

c diversified, such as by random, pseudorandom, or directed mutagenesis. Typically, CN VH and VL cassettes are diversified in or near the complementarity-determining Sregions (CDRS), often the third CDR, CDR3. Enzymatic inverse PCR mutagenesis C 10 has been shown to be a simple and reliable method for constructing relatively large libraries of scfv site-directed hybrids (Stemmer et al, 1993), as has error-prone PCR and chemical mutagenesis (Deng et al, 1994). Riechmann (Riechmann et al, 1993) showed semi-rational design of an antibody scfv fragment using site-directed randomization by degenerate oligonucleotide PCR and subsequent phage display of the resultant scfv hybrids. Barbas (Barbas et al, 1992) attempted to circumvent the problem of limited repertoire sizes resulting from using biased variable region sequences by randomizing the sequence in a synthetic CDR region of a human tetanus toxoid-binding Fab.

CDR randomization has the potential to create approximately 1 x 1020 CDRs for the heavy chain CDR3 alone, and a roughly similar number of variants of the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants. Taken individually or together, the combination possibilities of CDR randomization of heavy and/or light chains requires generating a prohibitive number of bacteriophage clones to produce a clone library representing all possible combinations, the vast majority of which will be non-binding. Generation of such large numbers of primary transformants is not feasible with current transformation technology and bacteriophage display systems. For example, Barbas (Barbas et al, 1992) only generated 5 x 10 7 transformants, which represents only a tiny fraction of the potential diversity of a library of thoroughly randomized CDRS.

177 i Despite these substantial limitations, bacteriophage display of scfv have already yielded a variety of useful antibodies and antibody fusion proteins. A bispecific single chain antibody has been shown to mediate efficient tumor cell lysis (Gruber et al, 1994). Intracellular expression of an anti-Rev scfv has been shown to oO 5 inhibit HIV-1 virus replication in vitro (Duan et al, 1994), and intracellular expression of an anti-p21rar, scfv has been shown to inhibit meiotic maturation of Xenopus oocytes (Biocca et al, 1993). Recombinant scfv which can be used to diagnose HIV infection have also been reported, demonstrating the diagnostic utility ofscfv (Lilley et al, 1994). Fusion proteins wherein an scFv is linked to a second polypeptide, such as a toxin or fibrinolytic activator protein, have also been reported (Holvost et al, 1992; Nicholls et al, 1993).

If it were possible to generate scfv libraries having broader antibody diversity and overcoming many of the limitations of conventional CDR mutagenesis and randomization methods which can cover only a very tiny fraction of the potential sequence combinations, the number and quality of scfv antibodies suitable for therapeutic and diagnostic use could be vastly improved. To address this, the in vitro and in vivo shuffling methods of the invention are used to recombine CDRs which have been obtained (typically via PCR amplification or cloning) from nucleic acids obtained from selected displayed antibodies. Such displayed antibodies can be displayed on cells, on bacteriophage particles, on polysomes, or any suitable antibody display system wherein the antibody is associated with its encoding nucleic acid(s). In a variation, the CDRs are initially obtained from mRNA (or cDNA) from antibody-producing cells plasma cells/splenocytes from an immunized wild-type mouse, a human, or a transgenic mouse capable of making a human antibody as in WO 92/03918, WO 93/12227, and WO 94/25585), including hybridomas derived therefrom.

Polynucleotide sequences selected in a first selection round (typically by affinity selection for displayed antibody binding to an antigen a ligand) by any of these methods are pooled and the pool(s) is/are shuffled by in vitro and/or in vivo recombination, especially shuffling of CDRs (typically shuffling heavy chain CDRs 178 with other heavy chain CDRs and light chain CDRs with other light chain CDRs) to produce a shuffled pool comprising a population of recombined selected polynucleotide sequences. The recombined selected polynucleotide sequences are expressed in a selection format as a displayed antibody and subjected to at least one subsequent selection round. The polynucleotide sequences selected in the subsequent selection round(s) can be used directly, sequenced, and/or subjected to n one or more additional rounds of shuffling and subsequent selection until an Cantibody of the desired binding affiity is obtained. Selected sequences can also be Sback-crossed with polynucleotide sequences encoding neutral antibody framework C 10 sequences having insubstantial functional effect on antigen binding), such as for example by back-crossing with a human variable region framework to produce human-like sequence antibodies. Generally, during back-crossing subsequent selection is applied to retain the property of binding to the predetermined antigen.

Alternatively, or in combination with the noted variations, the valency of the target epitope may be varied to control the average binding affinity of selected scfv library members. The target epitope can be bound to a surface or substrate at varying densities, such as by including a competitor epitope, by dilution, or by other method known to those in the art. A high density (valency) of predetermined epitope can be used to enrich for scfv library members which have relatively low affinity, whereas a low density (valency) can preferentially enrich for higher affinity scfv library members.

For generating diverse variable segments, a collection of synthetic oligonucleotides encoding random, pseudorandom, or a defined sequence keral set of peptide sequences can be inserted by ligation into a predetermined site a CDR). Similarly, the sequence diversity of one or more CDRs of the single-chain antibody cassette(s) can be expanded by mutating the CDR(s) with site-directed mutagenesis, CDR-replacement, and the like. The resultant DNA molecules can be propagated in a host for cloning and amplification prior to shuffling, or can be used directly may avoid loss of diversity which may occur upon propagation in a host cell) and the selected library members subsequently shuffled.

179 SDisplayed peptide/polynucleotide complexes (library members) which encode a variable segment peptide sequence of interest or a single-chain antibody of interest are selected from the library by an affinity enrichment technique. This is accomplished by means of a immobilized macromolecule or epitope specific for the 00 peptide sequence of interest, such as a receptor, other macromolecule, or other Cc epitope species. Repeating the affinity selection procedure provides an enrichment I of library members encoding the desired sequences, which may then be isolated for pooling and shuffling, for sequencing, and/or for further propagation and affinity N 10 enrichment.

The library members without the desired specificity are removed by washing.

The degree and stringency of washing required will be determined for each peptide sequence or single-chain antibody of interest and the immobilized predetermined macromolecule or epitope. A certain degree of control can be exerted over the binding characteristics of the nascent peptide/DNA complexes recovered by adjusting the conditions of the binding incubation and the subsequent washing. The temperature, pH, ionic strength, divalent cations concentration, and the volume and duration of the washing will select for nascent peptide/DNA complexes within particular ranges of affinity for the immobilized macromolecule. Selection based on slow dissociation rate, which is usually predictive of high affinity, is often the most practical route. This may be done either by continued incubation in the presence of a saturating amount of free predetermined macromolecule, or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated nascent peptide/DNA or peptide/RNA complex is prevented, and with increasing time, nascent peptide/DNA or peptide/RNA complexes of higher and higher affinity are recovered.

Additional modifications of the binding and washing procedures may be applied to find peptides with special characteristics. The affinities of some peptides are dependent on ionic strength or cation concentration. This is a useful characteristic for peptides that will be used in affinity purification of various 180 c proteins when gentle conditions for removing the protein from the peptides are required.

One variation involves the use of multiple binding targets (multiple epitope species, multiple receptor species), such that a scfv library can be simultaneously 00 screened for a multiplicity of scfv which have different binding specificities. Given

O

0C that the size of a scfv library often limits the diversity of potential scfv sequences, it is typically desirable to us scfv libraries of as large a size as possible. The time and 0 economic considerations of generating a number of very large polysome

C

10 scFv-display libraries can become prohibitive. To avoid this substantial problem, multiple predetermined epitope species (receptor species) can be concomitantly screened in a single library, or sequential screening against a number of epitope species can be used. In one variation, multiple target epitope species, each encoded on a separate bead (or subset of beads), can be mixed and incubated with a polysome-display scfv library under suitable binding conditions. The collection of beads, comprising multiple epitope species, can then be used to isolate, by affinity selection, scfv library members. Generally, subsequent affinity screening rounds can include the same mixture of beads, subsets thereof, or beads containing only one or two individual epitope species. This approach affords efficient screening, and is compatible with laboratory automation, batch processing, and high throughput screening methods.

A variety of techniques can be used in the present invention to diversify a peptide library or single-chain antibody library, or to diversify, prior to or concomitant with shuffling, around variable segment peptides found in early rounds of panning to have sufficient binding activity to the predetermined macromolecule or epitope. In one approach, the positive selected peptide/polynucleotide complexes (those identified in an early round of affinity enrichment) are sequenced to determine the identity of the active peptides. Oligonucleotides are then synthesized based on these active peptide sequences, employing a low level of all bases incorporated at each step to produce slight variations of the primary oligonucleotide sequences. This mixture of (slightly) degenerate oligonucleotides is then cloned into Z the variable segment sequences at the appropriate locations. This method produces Ssystematic, controlled variations of the starting peptide sequences, which can then be _shuffled. It requires, however, that individual positive nascent peptide/polynucleotide complexes be sequenced before mutagenesis, and thus is useful for expanding the diversity of small numbers of recovered complexes and selecting variants having higher binding affinity and/or higher binding specificity.

In a variation, mutagenic PCR amplification of positive selected peptide/polynucleotide complexes (especially of the variable region sequences, the Samplification products of which are shuffled in vitro and/or in vivo and one or more N 10 additional rounds of screening is done prior to sequencing. The same general approach can be employed with single-chain antibodies in order to expand the diversity and enhance the binding affinity/specificity, typically by diversifying CDRs or adjacent framework regions prior to or concomitant with shuffling. If desired, shuffling reactions can be spiked with mutagenic oligonucleotides capable of in vitro recombination with the selected library members can be included. Thus, mixtures of synthetic oligonucleotides and PCR produced polynucleotides (synthesized by error-prone or high-fidelity methods) can be added to the in vitro shuffling mix and be incorporated into resulting shuffled library members (shufflants).

The present invention of shuffling enables the generation of a vast library of CDR-variant single-chain antibodies. One way to generate such antibodies is to insert synthetic CDRs into the single-chain antibody and/or CDR randomization prior to or concomitant with shuffling. The sequences of the synthetic CDR cassettes are selected by referring to known sequence data of human CDR and are selected in the discretion of the practitioner according to the following guidelines: synthetic CDRs will have at least 40 percent positional sequence identity to known CDR.sequences, and preferably will have at least 50 to 70 percent positional sequence identity to known CDR sequences. For example, a collection of synthetic CDR sequences can be generated by synthesizing a collection of oligonucleotide sequences on the basis of naturally-occurring human CDR sequences listed in Kabat (Kabat et al, 1991); the pool of synthetic CDR sequences are calculated to 182 encode CDR peptide sequences having at least 40 percent sequence identity to at least one known naturally-occurring human CDR sequence. Alternatively, a collection of naturally-occurring CDR sequences may be compared to generate consensus sequences so that amino acids used at a residue position frequently in at least 5 percent of known CDR sequences) are incorporated into the synthetic 00 CDRs at the corresponding position(s). Typically, several 3 to about 50) known Cc CDR sequences are compared and observed natural sequence variations between the C known CDRs are tabulated, and a collection of oligonucleotides encoding CDR Speptide sequences encompassing all or most permutations of the observed natural C 10 sequence variations is synthesized. For example but not for limitation, if a collection of human VH CDR sequences have carboxy-terminal amino acids which are either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR oligonucleotide sequences are designed to allow the carboxy-terminal CDR residue to be any of these amino acids. In some embodiments, residues other than those which naturally-occur at a residue position in the collection of CDR sequences are incorporated: conservative amino acid substitutions are frequently incorporated and up to 5 residue positions may be varied to incorporate non-conservative amino acid substitutions as compared to known naturally-occurring CDR sequences. Such CDR sequences can be used in primary library members (prior to first round screening) and/or can be used to spike in vitro shuffling reactions of selected library member sequences. Construction of such pools of defined and/or degenerate sequences will be readily accomplished by those of ordinary skill in the art.

The collection of synthetic CDR sequences comprises at least one member that is not known to be a naturally-occurring CDR sequence. It is within the discretion of the practitioner to include or not include a portion of random or pseudorandom sequence corresponding to N region addition in the heavy chain CDR; the N region sequence ranges from 1 nucleotide to about 4 nucleotides occurring at V-D and D-J junctions. A collection of synthetic heavy chain CDR sequences comprises at least about 100 unique CDR sequences, typically at least about 1,000 unique CDR sequences, preferably at least about 10,000 unique CDR sequences, frequently more than 50,000 unique CDR sequences; however, usually 183 c not more than about 1 x 10 6 unique CDR sequences are included in the collection, Salthough occasionally 1 x 107 to I X 108 unique CDR sequences are present, especially if conservative amino acid substitutions are permitted at positions where the conservative amino acid substituent is not present or is rare less than 0.1 00 5 percent) in that position in naturally-occurring human CDRS. In general, the number of unique CDR sequences included in a library should not exceed the expected number of primary transformants in the library by more than a factor of \t Such single-chain antibodies generally bind of about at least I x 10 preferably 0with an affinity of about at least 5 x 107 M-1, more preferably with an affinity of at least 1 x 108 M-1 to 1 x 109 M-l or more, sometimes up to 1 x 1010 M-1 or more.

Frequently, the predetermined antigen is a human protein, such as for example a human cell surface antigen CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biological macromolecule thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, Lselectin), or nonhuman disease associated macromolecule bacterial LPS, virion capsid protein or envelope glycoprotein) and the like.

High affinity single-chain antibodies of the desired specificity can be engineered and expressed in a variety of systems. For example, scfv have been produced in plants (Firek et al. 1993) and can be readily made in prokaryotic systems (Owens and Young, 1994; Johnson and Bird, 1991). Furthermore, the single-chain antibodies can be used as a basis for constructing whole antibodies or various fragments thereof (Kettleborough et al, 1994). The variable region encoding sequence may be isolated by PCR amplification or subcloning) and spliced to a sequence encoding a desired human constant region to encode a human sequence antibody more suitable for human therapeutic uses where immunogenicity is preferably minimized. The polynucleotide(s) having the resultant fully human encoding sequence(s) can be expressed in a host cell from an expression vector in a mammalian cell) and purified for pharmaceutical formulation.

The DNA expression constructs will typically include an expression control DNA sequence operably linked to the coding sequences, including 184 naturally-associated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and 00 purification of the mutant' "engineered" antibodies.

0 CN As stated previously, the DNA sequences will be expressed in hosts after the Ssequences have been operably linked to an expression control sequence CN 10 positioned to ensure the transcription and translation of the structural gene). These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, USPN 4,704,362, which is incorporated herein by reference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissue cell culture may also be used to produce the polypeptides of the present invention (see Winnacker, 1987), which is incorporated herein by reference). Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, and myeloma cell lines, but preferably transformed Bcells or hybridomas. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, cytomegalovirus, SV40, Adenovirus, Bovine Papilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting sequences of between 10 to 300 185 bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 51 or 31 to the transcription unit. They are also effective if located within an intron or within the coding sequence itself. Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian 00 systems are also commonly used, such as the mouse immunoglobulin heavy chain c enhancer.

SMammalian expression vector systems will also typically include a selectable CN 10 marker gene. Examples of suitable markers include, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene or prokaryotic genes conferring drug resistance. The first two marker genes prefer the use of mutant cell lines that lack the ability to grow without the addition of thymidine to the growth medium.

Transformed cells can then be identified by their ability to grow on non-supplemented media. Examples of prokaryotic drug resistance genes useful as markers include genes conferring resistance to G418, mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, lipofection, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, and microinjection (see, generally, Sambrook et al, 1982 and 1989).

Once expressed, the antibodies, individual mutated immunoglobulin chains, mutated antibody fragments, and other immunoglobulin polypeptides of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, fraction column chromatography, gel electrophoresis and the like (see. generally, Scopes, 1982). Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically or 186 in developing and performing assay procedures, immunofluorescent stainings, and the like (see, generally, Lefkovits and Pernis, 1979 and 1981; Lefkovits, 1997).

The antibodies generated by the method of the present invention can be used for diagnosis and therapy. By way of illustration and not limitation, they can be used to treat cancer, autoimmune diseases, or viral infections. For treatment of

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Scancer, the antibodies will typically bind to an antigen expressed preferentially on C cancer cells, such as erbB-2, CEA, CD33, and many other antigens and binding members well known to those skilled in the art.

End-Selection This invention provides a method for selecting a subset ofpolynucleotides from a starting set of polynucleotides, which method is based on the ability to discriminate one or more selectable features (or selection markers) present anywhere in a working polynucleotide, so as to allow one to perform selection for (positive selection) &/or against (negative selection) each selectable polynucleotide. In a preferred aspect, a method is provided termed end-selection, which method is based on the use of a selection marker located in part or entirely in a terminal region of a selectable polynucleotide, and such a selection marker may be termed an "endselection marker".

End-selection may be based on detection of naturally occurring sequences or on detection of sequences introduced experimentally (including by any mutagenesis procedure mentioned herein and not mentioned herein) or on both, even within the same polynucleotide. An end-selection marker can be a structural selection marker or a functional selection marker or both a structural and a functional selection marker. An end-selection marker may be comprised of a polynucleotide sequence or of a polypeptide sequence or of any chemical structure or of any biological or biochemical tag, including markers that can be selected using methods based on the detection of radioactivity, of enzymatic activity, of fluorescence, of any optical feature, of a magnetic property using magnetic beads), of immunoreactivity, and of hybridization.

187 rEnd-selection may be applied in combination with any method serviceable for performing mutagenesis. Such mutagenesis methods include, but are not limited to, methods described herein (supra and infra). Such methods include, by way of 0 5 non-limiting exemplification, any method that may be referred herein or by others in the art by any of the following terms: "saturation mutagenesis", "shuffling", "recombination", "re-assembly", "error-prone PCR", "assembly PCR", "sexual PCR", "crossover PCR", "oligonucleotide primer-directed mutagenesis", "recursive exponential) ensemble mutagenesis (see Arkin and Youvan, 1992)", "cassette mutagenesis", "in vivo mutagenesis", and "in vitro mutagenesis". Moreover, endselection may be performed on molecules produced by any mutagenesis &/or amplification method (see, Arnold, 1993; Caldwell and Joyce, 1992; Stemmer, 1994; following which method it is desirable to select for (including to screen for the presence of) desirable progeny molecules.

In addition, end-selection may be applied to a polynucleotide apart from any mutagenesis method. In a preferred embodiment, end-selection, as provided herein, can be used in order to facilitate a cloning step, such as a step of ligation to another polynucleotide (including ligation to a vector). This invention thus provides for endselection as a serviceable means to facilitate library construction, selection &/or enrichment for desirable polynucleotides, and cloning in general.

In a particularly preferred embodiment, end-selection can be based on (positive) selection for a polynucleotide; alternatively end-selection can be based on (negative) selection against a polynucleotide; and alternatively still, end-selection can be based on both (positive) selection for, and on (negative) selection against, a polynucleotide. End-selection, along with other methods of selection &/or screening, can be performed in an iterative fashion, with any combination of like or unlike selection &/or screening methods and serviceable mutagenesis methods, all of which can be performed in an iterative fashion and in any order, combination, and permutation.

188 It is also appreciated that, according to one embodiment of this invention, Send-selection may also be used to select a polynucleotide is at least in part: circular a plasmid or any other circular vector or any other polynucleotide that is partly circular), &/or branched, &/or modified or substituted with any chemical group or moiety. In accord with this embodiment, a polynucleotide may be a circular oO molecule comprised of an intermediate or central region, which region is flanked on c a 5' side by a 5' flanking region (which, for the purpose of end-selection, serves in like manner to a 5' terminal region of a non-circular polynucleotide) and on a 3' side Sby a 3' terminal region (which, for the purpose of end-selection, serves in like N 10 manner to a 3' terminal region of a non-circular polynucleotide). As used in this non-limiting exemplification, there may be sequence overlap between any two regions or even among all three regions.

In one non-limiting aspect of this invention, end-selection of a linear polynucleotide is performed using a general approach based on the presence of at least one end-selection marker located at or near a polynucleotide end or terminus (that can be either a 5' end or a 3' end). In one particular non-limiting exemplification, end-selection is based on selection for a specific sequence at or near a terminus such as, but not limited to, a sequence recognized by an enzyme that recognizes a polynucleotide sequence. An enzyme that recognizes and catalyzes a chemical modification of a polynucleotide is referred to herein as a polynucleotideacting enzyme. In a preferred embodiment, serviceable polynucleotide-acting enzymes are exemplified non-exclusively by enzymes with polynucleotide-cleaving activity, enzymes with polynucleotide-methylating activity, enzymes with polynucleotide-ligating activity, and enzymes with a plurality of distinguishable enzymatic activities (including non-exclusively, both polynucleotide-cleaving activity and polynucleotide-ligating activity).

Relevant polynucleotide-acting enzymes thus also include any commercially available or non-commercially available polynucleotide endonucleases and their companion methylases including those catalogued at the website http://www.neb.com/rebase, and those mentioned in the following cited reference 189 (c (Roberts and Macelis, 1996). Preferred polynucleotide endonucleases include -but are not limited to type II restriction enzymes (including type IIS), and include enzymes that cleave both strands of a double stranded polynucleotide Not I, which cleaves both strands at 5'...GC/GGCCGC...3') and enzymes that cleave only OC 5 one strand of a double stranded polynucleotide, i.e. enzymes that have polynucleotide-nicking activity, N. BstNB I, which cleaves only one strand at Relevant polynucleotide-acting enzymes also include i type III restriction enzymes.

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It is appreciated that relevant polynucleotide-acting enzymes also include any enzymes that may be developed in the future, though currently unavailable, that are serviceable for generating a ligation compatible end, preferably a sticky end, in a polynucleotide.

In one preferred exemplification, a serviceable selection marker is a restriction site in a polynucleotide that allows a corresponding type II (or type IIS) restriction enzyme to cleave an end of the polynucleotide so as to provide a ligatable end (including a blunt end or alternatively a sticky end with at least a one base overhang) that is serviceable for a desirable ligation reaction without cleaving the polynucleotide internally in a manner that destroys a desired internal sequence in the polynucleotide. Thus it is provided that, among relevant restriction sites, those sites that do not occur internally that do not occur apart from the termini) in a specific working polynucleotide are preferred when the use of a corresponding restriction enzyme(s) is not intended to cut the working polynucleotide internally.

This allows one to perform restriction digestion reactions to completion or to near completion without incurring unwanted internal cleavage in a working polynucleotide.

According to a preferred aspect, it is thus preferable to use restriction sites that are not contained, or alternatively that are not expected to be contained, or alternatively that unlikely to be contained when sequence information regarding a working polynucleotide is incomplete) internally in a polynucleotide to 190 be subjected to end-selection. In accordance with this aspect, it is appreciated that restriction sites that occur relatively infrequently are usually preferred over those _that occur more frequently. On the other hand it is also appreciated that there are occasions where internal cleavage of a polypeptide is desired, e.g. to achieve recombination or other mutagenic procedures along with end-selection.

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0 In accord with this invention, it is also appreciated that methods (e.g.

N mutagenesis methods) can be used to remove unwanted internal restriction sites. It Sis also appreciated that a partial digestion reaction a digestion reaction that C 10 proceeds to partial completion) can be used to achieve digestion at a recognition site in a terminal region while sparing a susceptible restriction site that occurs internally in a polynucleotide and that is recognized by the same enzyme. In one aspect, partial digest are useful because it is appreciated that certain enzymes show preferential cleavage of the same recognition sequence depending on the location and environment in which the recognition sequence occurs. For example, it is appreciated that, while lambda DNA has 5 EcoR I sites, cleavage of the site nearest to the right terminus has been reported to occur 10 times faster than the sites in the middle of the molecule. Also, for example, it has been reported that, while Sac II has four sites on lambda DNA, the three clustered centrally in lambda are cleaved times faster than the remaining site near the terminus (at nucleotide 40,386).

Summarily, site preferences have been reported for various enzymes by many investigators Thomas and Davis, 1975; Forsblum et al, 1976; Nath and Azzolina, 1981; Brown and Smith, 1977; Gingeras and Brooks, 1983; Kriiger et al, 1988; Conrad and Topal, 1989; Oiler et al, 1991; Topal, 1991; and Pein, 1991; to name but a few). It is appreciated that any empirical observations as well as any mechanistic understandings of site preferences by any serviceable polynucleotideacting enzymes, whether currently available or to be procured in the future, may be serviceable in end-selection according to this invention.

It is also appreciated that protection methods can be used to selectively protect specified restriction sites internal sites) against unwanted digestion by enzymes that would otherwise cut a working polypeptide in response to the presence 191 C1 of those sites; and that such protection methods include modifications such as methylations and base substitutions U instead ofT) that inhibit an unwanted enzyme activity. It is appreciated that there are limited numbers of available restriction enzymes that are rare enough having very long recognition oO 5 sequences) to create large megabase-long) restriction fragments, and that protection approaches by methylation) are serviceable for increasing the rarity of enzyme cleavage sites. The use of M.Fnu II (mCGCG) to increase the apparent N rarity of Not I approximately twofold is but one example among many (Qiang et al, 1990; Nelson et al, 1984; Maxam and Gilbert, 1980; Raleigh and Wilson, 1986).

According to a preferred aspect of this invention, it is provided that, in general, the use of rare restriction sites is preferred. It is appreciated that, in general, the frequency of occurrence of a restriction site is determined by the number of nucleotides contained therein, as well as by the ambiguity of the base requirements contained therein. Thus, in a non-limiting exemplification, it is appreciated that, in general, a restriction site composed of, for example, 8 specific nucleotides the Not I site or GC/GGCCGC, with an estimated relative occurrence of 1 in 48, i.e. 1 in 65,536, random 8-mers) is relatively more infrequent than one composed of, for example, 6 nucleotides the Sma I site or CCC/GGG, having an estimated relative occurrence of 1 in 46, i.e. 1 in 4,096, random 6-mers), which in turn is relatively more infrequent than one composed of, for example, 4 nucleotides (e.g.

the Msp I site or C/CGG, having an estimated relative occurrence of 1 in 44, i.e. 1 in 256, random 4-mers). Moreover, in another non-limiting exemplification, it is appreciated that, in general, a restriction site having no ambiguous (but only specific) base requirements the Fin I site or GTCCC, having an estimated relative occurrence of 1 in 45, i.e. I in 1024, random 5-mers) is relatively more infrequent than one having an ambiguous W (where W A or T) base requirement the Ava II site or G/GWCC, having an estimated relative occurrence of 1 in 4x4x2x4x4 i.e. 1 in 512 random 5-mers), which in turn is relatively more infrequent than one having an ambiguous N (where N A or C or G or T) base requirement the Asu I site or G/GNCC, having an estimated relative occurrence of 1 in4x4xlx4x4, i.e. 1 in 256 random 5-mers). These relative occurrences are 192 Q) considered general estimates for actual polynucleotides, because it is appreciated that specific nucleotide bases (not to mention specific nucleotide sequences) occur with dissimilar frequencies in specific polynucleotides, in specific species of organisms, and in specific groupings of organisms. For example, it is appreciated that the G+C contents of different species of organisms are often very different oO Sand wide ranging.

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CN The use of relatively more infrequent restriction sites as a selection marker 0include in a non-limiting fashion preferably those sites composed at least a 4 nucleotide sequence, more preferably those composed at least a 5 nucleotide sequence, more preferably still those composed at least a 6 nucleotide sequence (e.g.

the BamH I site or G/GATCC, the Bgl II site or A/GATCT, the Pst I site or CTGCA/G, and the Xba I site or T/CTAGA), more preferably still those composed at least a 7 nucleotide sequence, more preferably still those composed of an 8 nucleotide sequence nucleotide sequence the Asc I site or GG/CGCGCC, the Not I site or GC/GGCCGC, the Pac I site or TTAAT/TAA, the Pme I site or GTTT/AAAC, the SrfI site or GCCC/GGGC, the Sse838 I site or CCTGCA/GG, and the Swa I site or ATTT/AAAT), more preferably still those composed of a 9 nucleotide sequence, and even more preferably still those composed of at least a nucleotide sequence the BspG I site or CG/CGCTGGAC). It is further appreciated that some restriction sites for class IIS enzymes) are comprised of a portion of relatively high specificity a portion containing a principal determinant of the frequency of occurrence of the restriction site) and a portion of relatively low specificity; and that a site of cleavage may or may not be contained within a portion of relatively low specificity. For example, in the Eco57 I site or CTGAAG(16/14), there is a portion of relatively high specificity the CTGAAG portion) and a portion of relatively low specificity the NI6 sequence) that contains a site of cleavage.

In another preferred embodiment of this invention, a serviceable endselection marker is a terminal sequence that is recognized by a polynucleotide-acting enzyme that recognizes a specific polynucleotide sequence. In a preferred aspect of 193 this invention, serviceable polynucleotide-acting enzymes also include other enzymes in addition to classic type II restriction enzymes. According to this Spreferred aspect of this invention, serviceable polynucleotide-acting enzymes also include gyrases, helicases, recombinases, relaxases, and any enzymes related thereto.

00 M Among preferred examples are topoisomerases (which have been categorized N by some as a subset of the gyrases) and any other enzymes that have polynucleotide- O cleaving activity (including preferably polynucleotide-nicking activity) &/or CN 10 polynucleotide-ligating activity. Among preferred topoisomerase enzymes are topoisomerase I enzymes, which is available from many commercial sources (Epicentre Technologies, Madison, WI; Invitrogen, Carlsbad, CA; Life Technologies, Gathesburg, MD) and conceivably even more private sources. It is appreciated that similar enzymes may be developed in the future that are serviceable for end-selection as provided herein. A particularly preferred topoisomerase I enzyme is a topoisomerase I enzyme of vaccinia virus origin, that has a specific recognition sequence and has both polynucleotide-nicking activity and polynucleotide-ligating activity. Due to the specific nicking-activity of this enzyme (cleavage of one strand), internal recognition sites are not prone to polynucleotide destruction resulting from the nicking activity (but rather remain annealed) at a temperature that causes denaturation of a terminal site that has been nicked. Thus for use in end-selection, it is preferable that a nicking site for topoisomerase-based end-selection be no more than 100 nucleotides from a terminus, more preferably no more than 50 nucleotides from a terminus, more preferably still no more than 25 nucloetides from a terminus, even more preferably still no more than 20 nucleotides from a terminus, even more preferably still no more than 15 nucleotides from a terminus, even more preferably still no more than nucleotides from a terminus, even more preferably still no more than 8 nucleotides from a terminus, even more preferably still no more than 6 nucleotides from a terminus, and even more preferably still no more than 4 nucleotides from a terminus.

SIn a particularly preferred exemplification that is non-limiting yet clearly illustrative, it is appreciated that when a nicking site for topoisomerase-based endselection is 4 nucleotides from a terminus, nicking produces a single stranded oligo of 4 bases (in a terminal region) that can be denatured from its complementary strand in an end-selectable polynucleotide; this provides a sticky end (comprised of

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S4 bases) in a polynucleotide that is serviceable for an ensuing ligation reaction. To c accomplish ligation to a cloning vector (preferably an expression vector), compatible C sticky ends can be generated in a cloning vector by any means including by Srestriction enzyme-based means. The terminal nucleotides (comprised of 4 terminal C 10 bases in this specific example) in an end-selectable polynucleotide terminus are thus wisely chosen to provide compatibility with a sticky end generated in a cloning vector to which the polynucleotide is to be ligated.

On the other hand, internal nicking of an end-selectable polynucleotide, e.g.

500 bases from a terminus, produces a single stranded oligo of 500 bases that is not easily denatured from its complementary strand, but rather is serviceable for repair by the same topoisomerase enzyme that produced the nick).

This invention thus provides a method e.g. that is vaccinia topoisomerasebased &/or type II (or IIS) restriction endonuclease-based &/or type III restriction endonuclease-based &/or nicking enzyme-based using N. BstNB I) for producing a sticky end in a working polynucleotide, which end is ligation compatible, and which end can be comprised of at least a 1 base overhang.

Preferably such a sticky end is comprised of at least a 2-base overhang, more preferably such a sticky end is comprised of at least a 3-base overhang, more preferably still such a sticky end is comprised of at least a 4-base overhang, even more preferably still such a sticky end is comprised of at least a 5-base overhang, even more preferably still such a sticky end is comprised of at least a 6-base overhang. Such a sticky end may also be comprised of at least a 7-base overhang, or at least an 8-base overhang, or at least a 9-base overhang, or at least a overhang, or at least 15-base overhang, or at least a 20-base overhang, or at least a 195 overhang, or at least a 30-base overhang. These overhangs can be comprised of any bases, including A, C, G, or T.

It is appreciated that sticky end overhangs introduced using topoisomerase or a nicking enzyme using N. BstNB I) can be designed to be unique in a ligation oO environment, so as to prevent unwanted fragment reassemblies, such as self-

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r dimerizations and other unwanted concatamerizations.

SAccording to one aspect of this invention, a plurality of sequences (which C 10 may but do not necessarily overlap) can be introduced into a terminal region of an end-selectable polynucleotide by the use of an oligo in a polymerase-based reaction.

In a relevant, but by no means limiting example, such an oligo can be used to provide a preferred 5' terminal region that is serviceable for topoisomerase I-based end-selection, which oligo is comprised of: a 1-10 base sequence that is convertible into a sticky end (preferably by a vaccinia topoisomerase a ribosome binding site and "RBS", that is preferably serviceable for expression cloning), and optional linker sequence followed by an ATG start site and a template-specific sequence of 0- 100 bases (to facilitate annealment to the template in the a polymerase-based reaction). Thus, according to this example, a serviceable oligo (which may be termed a forward primer) can have the sequence: 5'[terminal sequence (N)i.

lo][topoisomerase I site RBS AAGGGAGGAG][linker (N)I-.oo][start codon and template-specific sequence ATG(N)o- 1 oo]3'.

Analogously, in a relevant, but by no means limiting example, an oligo can be used to provide a preferred 3' terminal region that is serviceable for topoisomerase I-based end-selection, which oligo is comprised of: a 1-10 base sequence that is convertible into a sticky end (preferably by a vaccinia topoisomerase and optional linker sequence followed by a template-specific sequence of 0-100 bases (to facilitate annealment to the template in the a polymerase-based reaction). Thus, according to this example, a serviceable oligo (which may be termed a reverse primer) can have the sequence: 196 sequence (N) 1 1 o][topoisomerase I site AAGGG][linker (N),l.oo][templatespecific sequence (N)o-oo]3'.

It is appreciated that, end-selection can be used to distinguish and separate 5 parental template molecules to be subjected to mutagenesis) from progeny oO molecules generated by mutagenesis). For example, a first set of primers, lacking in C a topoisomerase I recognition site, can be used to modify the terminal regions of the parental molecules in polymerase-based amplification). A different second set of Sprimers having a topoisomerase I recognition site) can then be used to generate C 10 mutated progeny molecules using any polynucleotide chimerization method, such as interrupted synthesis, template-switching polymerase-based amplification, or interrupted synthesis; or using saturation mutagenesis; or using any other method for introducing a topoisomerase I recognition site into a mutagenized progeny molecule as disclosed herein) from the amplified template molecules. The use of topoisomerase I-based endselection can then facilitate, not only discernment, but selective topoisomerase I-based ligation of the desired progeny molecules.

Annealment of a second set of primers to thusly amplified parental molecules can be facilitated by including sequences in a first set of primers primers used for amplifying a set parental molecules) that are similar to a toposiomerase I recognition site, yet different enough to prevent functional toposiomerase I enzyme recognition. For example, sequences that diverge from the AAGGG site by anywhere from 1 base to all bases can be incorporated into a first set of primers (to be used for amplifying the parental templates prior to subjection to mutagenesis). In a specific, but non-limiting aspect, it is thus provided that a parental molecule can be amplified using the following exemplary but by no means limiting set of forward and reverse primers: Forward Primer: 5' CTAGAAGAGAGGAGAAAACCATG(N)to-too and Reverse Primer: 5' GATCAAAGGCGCGCCTGCAGG(N)lo-oo 3' According to this specific example of a first set of primers, (N)to-loo represents preferably a 10 to 100 nucleotide-long template-specific sequence, more preferably a 197 Sto 50 nucleotide-long template-specific sequence, more preferably still a 10 to Snucleotide-long template-specific sequence, and even more preferably still a 15 to nucleotide-long template-specific sequence.

According to a specific, but non-limiting aspect, it is thus provided that, after this amplification (using a disclosed first set of primers lacking in a true topoisomerase I recognition site), amplified parental molecules can then be subjected to mutagenesis N using one or more sets of forward and reverse primers that do have a true topoisomerase SI recognition site. In a specific, but non-limiting aspect, it is thus provided that a parental N 10 molecule can be used as templates for the generation of a mutagenized progeny molecule using the following exemplary but by no means limiting second set of forward and reverse primers: Forward Primer: 5' CTAGAAGGGAGGAGAAAACCATG 3' Reverse Primer. 5' GATCAAAGGCGCGCCTGCAGG 3' (contains Asc I recognition sequence) It is appreciated that any number of different primers sets not specifically mentioned can be used as first, second, or subsequent sets of primers for end-selection consistent with this invention. Notice that type II restriction enzyme sites can be incorporated an Asc I site in the above example). It is provided that, in addition to the other sequences mentioned, the experimentalist can incorporate one or more N,N,G/T triplets into a serviceable primer in order to subject a working polynucleotide to saturation mutagenesis. Summarily, use of a second and/or subsequent set of primers can achieve dual goals of introducing a topoisomerase I site and of generating mutations in a progeny polynucleotide.

Thus, according to one use provided, a serviceable end-selection marker is an enzyme recognition site that allows an enzyme to cleave (including nick) a polynucleotide at a specified site, to produce a ligation-compatible end upon denaturation of a generated single stranded oligo. Ligation of the produced polynucleotide end can then be accomplished by the same enzyme in the case 198 Sof vaccinia virus toposiomerase or alternatively with the use of a different enzyme. According to one aspect of this invention, any serviceable end-selection markers, whether like two vaccinia virus toposiomerase I recognition sites) or unlike a class II restriction enzyme recognition site and a vaccinia virus toposiomerase I recognition site) can be used in combination to select a oO Spolynucleotide. Each selectable polynucleotide can thus have one or more end-

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Cr selection markers, and they can be like or unlike end-selection markers. In a CN particular aspect, a plurality of end-selection markers can be located on one end of a 0polynucleotide and can have overlapping sequences with each other.

C It is important to emphasize that any number of enzymes, whether currently in existence or to be developed, can be serviceable in end-selection according to this invention. For example, in a particular aspect of this invention, a nicking enzyme N. BstNB I, which cleaves only one strand at 5'...GAGTCNNNN/N...3') can be used in conjunction with a source of polynucleotide-ligating activity in order to achieve end-selection. According to this embodiment, a recognition site for N. BstN BI instead of a recognition site for topoisomerase I should be incorported into an end-selectable polynucleotide (whether end-selection is used for selection of a mutagenized progeny molecule or whether end-selection is used apart from any mutagenesis procedure).

It is appreciated that the instantly disclosed end-selection approach using topoisomerase-based nicking and ligation has several advantages over previously available selection methods. In sum, this approach allows one to achieve direction cloning (including expression cloning). Specifically, this approach can be used for the achievement of: direct ligation without subjection to a classic restrictionpurification-ligation reaction, that is susceptible to a multitude of potential problems from an initial restriction reaction to a ligation reaction dependent on the use ofT4 DNA ligase); separation of progeny molecules from original template molecules original template molecules lack topoisomerase I sites that not introduced until after mutagenesis), obviation of the need for size separation steps by gel chromatography or by other electrophoretic means or by the use of size-exclusion 199 membranes), preservation of internal sequences (even when topoisomerase I sites are present), obviation of concerns about unsuccessful ligation reactions (e.g.

dependent on the use of T4 DNA ligase, particularly in the presence of unwanted residual restriction enzyme activity), and facilitated expression cloning (including 5 obviation of frame shift concerns). Concerns about unwanted restriction enzyme- 00 based cleavages especially at internal restriction sites (or even at often n unpredictable sites of unwanted star activity) in a working polynucleotide that are potential sites of destruction of a working polynucleotide can also be obviated by the Sinstantly disclosed end-selection approach using topoisomerase-based nicking and C 10 ligation.

Two-Hybrid Based Screening Assays Shuffling can also be used to recombinatorially diversify a pool of selected library members obtained by screening a two-hybrid screening system to identify library members which bind a predetermined polypeptide sequence. The selected library members are pooled and shuffled by in vitro and/or in vivo recombination.

The shuffled pool can then be screened in a yeast two hybrid system to select library members which bind said predetermined polypeptide sequence and SH2 domain) or which bind an alternate predetermined polypeptide sequence an SH2 domain from another protein species).

An approach to identifying polypeptide sequences which bind to a predetermined polypeptide sequence has been to use a so-called "two-hybrid" system wherein the predetermined polypeptide sequence is present in a fusion protein (Chien et al, 1991). This approach identifies protein-protein interactions in vivo through reconstitution of a transcriptional activator (Fields and Song, 1989), the yeast Gal4 transcription protein. Typically, the method is based on the properties of the yeast Gal4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. Polynucleotides encoding two hybrid proteins, one consisting of the yeast Gal4 DNA-binding domain fused to a 200 Spolypeptide sequence of a known protein and the other consisting of the Gal4 activation domain fused to a polypeptide sequence of a second protein, are constructed and introduced into a yeast host cell. Intermolecular binding between the two fusion proteins reconstitutes the Gal4 DNA-binding domain with the Gal4 activation domain, which leads to the transcriptional activation of a reporter gene

OO

lacz, HIS3) which is operably linked to a Gal4 binding site. Typically, the

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Cc two-hybrid method is used to identify novel polypeptide sequences which interact 0 C with a known protein (Silver and Hunt, 1993; Durfee et al, 1993; Yang et al, 1992; 0 Luban et al, 1993; Hardy et al, 1992; Bartel et al, 1993; and Vojtek et al, 1993).

C1 10 However, variations of the two-hybrid method have been used to identify mutations of a known protein that affect its binding to a second known protein (Li and Fields, 1993; Lalo et al, 1993; Jackson et al, 1993; and Madura et al, 1993). Two-hybrid systems have also been used to identify interacting structural domains of two known proteins (Bardwell et al, 1993; Chakrabarty et al, 1992; Staudinger et al, 1993; and Milne and Weaver 1993) or domains responsible for oligomerization of a single protein (Iwabuchi et al, 1993; Bogerd et al, 1993). Variations of two-hybrid systems have been used to study the in vivo activity of a proteolytic enzyme (Dasmahapatra et al, 1992). Alternatively, an E. coli/BCCP interactive screening system (Germino et al, 1993; Guarente, 1993) can be used to identify interacting protein sequences protein sequences which heterodimerize or form higher order heteromultimers). Sequences selected by a two-hybrid system can be pooled and shuffled and introduced into a two-hybrid system for one or more subsequent rounds of screening to identify polypeptide sequences which bind to the hybrid containing the predetermined binding sequence. The sequences thus identified can be compared to identify consensus sequence(s) and consensus sequence kernals.

In general, standard techniques of recombination DNA technology are described in various publications Sambrook et al, 1989; Ausubel et al, 1987; and Berger and Kimmel, 1987), each of which is incorporated herein in its entirety by reference. Polynucleotide modifying enzymes were used according to the manufacturer's recommendations. Oligonucleotides were synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizer using ABI chemicals. If desired, PCR 201 Samplimers for amplifying a predetermined DNA sequence may be selected at the discretion of the practitioner.

One microgram samples of template DNA are obtained and treated with U.V.

5 light to cause the formation of dimers, including TT dimers, particularly purine oO dimers. U.V. exposure is limited so that only a few photoproducts are generated per Sgene on the template DNA sample. Multiple samples are treated with U.V. light for varying periods of time to obtain template DNA samples with varying numbers of Sdimers from U.V. exposure.

I A random priming kit which utilizes a non-proofreading polymease (for example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems) is utilized to generate different size polynucleotides by priming at random sites on templates which are prepared by U.V. light (as described above) and extending along the templates. The priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers. The dimers formed by U.V. exposure serve as a roadblock for the extension by the non-proofreading polymerase. Thus, a pool of random size polynucleotides is present after extension with the random primers is finished.

The present invention is further directed to a method for generating a selected mutant polynucleotide sequence (or a population of selected polynucleotide sequences) typically in the form of amplified and/or cloned polynucleotides, whereby the selected polynucleotide sequences(s) possess at least one desired phenotypic characteristic encodes a polypeptide, promotes transcription of linked polynucleotides, binds a protein, and the like) which can be selected for. One method for identifying hybrid polypeptides that possess a desired structure or functional property, such as binding to a predetermined biological macromolecule a receptor), involves the screening of a large library of polypeptides for individual library members which possess the desired structure or functional property conferred by the amino acid sequence of the polypeptide.

202 Q- In one embodiment, the present invention provides a method for generating libraries of displayed polypeptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening. The method comprises obtaining a first plurality of selected library members comprising a displayed polypeptide or displayed antibody and an associated polynucleotide encoding said displayed

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Spolypeptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of

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C substantially identical sequences, optimally introducing mutations into said 0 polynucleotides or copies, pooling the polynucleotides or copies, producing C 10 smaller or shorter polynucleotides by interrupting a random or particularized priming and synthesis process or an amplification process, and performing amplification, preferably PCR amplification, and optionally mutagenesis to homologously recombine the newly synthesized polynucleotides.

It is a particularly preferred object of the invention to provide a process for producing hybrid polynucleotides which express a useful hybrid polypeptide by a series of steps comprising: producing polynucleotides by interrupting a polynucleotide amplification or synthesis process with a means for blocking or interrupting the amplification or synthesis process and thus providing a plurality of smaller or shorter polynucleotides due to the replication of the polynucleotide being in various stages of completion; adding to the resultant population of single- or double-stranded polynucleotides one or more single- or double-stranded oligonucleotides, wherein said added oligonucleotides comprise an area of identity in an area of heterology to one or more of the single- or double-stranded polynucleotides of the population; denaturing the resulting single- or double-stranded oligonucleotides to produce a mixture of single-stranded polynucleotides, optionally separating the shorter or smaller polynucleotides into pools of polynucleotides having various lengths and further optionally subjecting said polynucleotides to a PCR procedure to amplify one or more oligonucleotides comprised by at least one of said polynucleotide pools; 203 incubating a plurality of said polynucleotides or at least one pool of said polynucleotides with a polymerase under conditions which result in annealing of said single-stranded polynucleotides at regions of identity between the singlestranded polynucleotides and thus forming of a mutagenized double-stranded polynucleotide chain; 00 optionally repeating steps and

O

m expressing at least one hybrid polypeptide from said polynucleotide N chain, or chains; and S(g) screening said at least one hybrid polypeptide for a useful activity.

C 10 In a preferred aspect of the invention, the means for blocking or interrupting the amplification or synthesis process is by utilization ofU.V. light, DNA adducts, DNA binding proteins.

In one embodiment of the invention, the DNA adducts, or polynucleotides comprising the DNA adducts, are removed from the polynucleotides or polynucleotide pool, such as by a process including heating the solution comprising the DNA fragments prior to further processing.

Having thus disclosed exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures are exemplary only and that various other alternatives, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent.

The following examples are to be considered illustrative and thus are not limiting of the remainder of the disclosure in any way whatsoever.

204 Q- Example 1 Generation of Random Size Polynucleotides Using U.V. Induced Photoproducts One microgram samples of template DNA are obtained and treated with U.V.

light to cause the formation of dimers, including TT dimers, particularly purine dimers. U.V. exposure is limited so that only a few photoproducts are generated per 00 gene on the template DNA sample. Multiple samples are treated with U.V. light for Svarying periods of time to obtain template DNA samples with varying numbers of C dimers from U.V. exposure.

A random priming kit which utilizes a non-proofreading polymease (for C 10 example, Prime-It II Random Primer Labeling kit by Stratagene Cloning Systems) is utilized to generate different size polynucleotides by priming at random sites on templates which are prepared by U.V. light (as described above) and extending along the templates. The priming protocols such as described in the Prime-It II Random Primer Labeling kit may be utilized to extend the primers. The dimers formed by' U.V. exposure serve as a roadblock for the extension by the non-proofreading polymerase. Thus, a pool of random size polynucleotides is present after extension with the random primers is finished.

205 SExample 2 Isolation of Random Size Polvnucleotides Polynucleotides of interest which are generated according to Example 1 are are gel isolated on a 1.5% agarose gel. Polynucleotides in the 100-300 bp range are cut out of the gel and 3 volumes of 6 M Nal is added to the gel slice. The mixture is 00 incubated at 50 *C for 10 minutes and 10 j1 of glass milk (Bio 101) is added. The

O

mC mixture is spun for I minute and the supernatant is decanted. The pellet is washed (N with 500 l1 of Column Wash (Column Wash is 50% ethanol, 10mM Tris-HCl pH 100 mM NaCI and 2.5 mM EDTA) and spin for 1 minute, after which the CN 10 supernatant is decanted. The washing, spinning and decanting steps are then repeated. The glass milk pellet is resuspended in 20p1 of H 2 0 and spun for 1 minute. DNA remains in the aqueous phase.

206 Example 3 Shuffling of Isolated Random Size 100-300bp Polynucleotides The 100-300 bp polynucleotides obtained in Example 2 are recombined in an annealing mixture (0.2 mM each dNTP, 2.2 mM MgCl 2 50 mM KC1, 10 mM Tris- HCI ph 8.8, 0.1% Triton X-100, 0.3 jt; Taq DNA polymerase, 50 pl total volume) 00 without adding primers. A Robocycler by Stratagene was used for the annealing n step with the following program: 95 OC for 30 seconds, 25-50 cycles of [95 °C for seconds, 50 60 *C (preferably 58 OC) for 30 seconds, and 72 OC for 30 seconds] and 5 minutes at 72 OC. Thus, the 100-300 bp polynucleotides combine to yield double-stranded polynucleotides having a longer sequence. After separating out the reassembled double-stranded polynucleotides and denaturing them to form single stranded polynucleotides, the cycling is optionally again repeated with some samples utilizing the single strands as template and primer DNA and other samples utilizing random primers in addition to the single strands.

207 Example 4 Screening of Polypeptides from Shuffled Polynucleotides The polynucleotides of Example 3 are separated and polypeptides are expressed therefrom. The original template DNA is utilized as a comparative control by obtaining comparative polypeptides therefrom. The polypeptides 00oO obtained from the shuffled polynucleotides of Example 3 are screened for the c activity of the polypeptides obtained from the original template and compared with 1 the activity levels of the control. The shuffled polynucleotides coding for interesting Spolypeptides discovered during screening are compared further for secondary C 10 desirable traits. Some shuffled polynucleotides corresponding to less interesting screened polypeptides are subjected to reshuffling.

208 Q Example Directed Evolution an Enzyme by Saturation Mutagenesis SSite-Saturation Mutagenesis: To accomplish site-saturation mutagenesis every residue (316) of a dehalogenase enzyme was converted into all 20 amino acids by 00 site directed mutagenesis using 32-fold degenerate oligonucleotide primers, as 0 follows: i 1. A culture of the dehalogenase expression construct was grown and a preparation of the plasmid was made 2. Primers were made to randomize each codon they have the common structure

X

2 oNN(G/T)X 2 0 3. A reaction mix of 25 ul was prepared containing -50 ng ofplasmid template, 125 ng of each primer, IX native Pfu buffer, 200 uM each dNTP and 2.5 U native Pfu DNA polymerase 4. The reaction was cycled in a Robo96 Gradient Cycler as follows: Initial denaturation at 95C for 1 min cycles of 95 0 C for 45 sec, 53°C for 1 min and 72 0 C for 11 min Final elongation step of 72C for 10 min 5. The reaction mix was digested with 10 U of DpnI at 37 0 C for 1 hour to digest the methylated template DNA 6. Two ul of the reaction mix were used to transform 50 ul ofXLl-Blue MRF' cells and the entire transformation mix was plated on a large LB-Amp-Met plate yielding 200-1000 colonies 7. Individual colonies were toothpicked into the wells of 96-well microtiter plates containing LB-Amp-IPTG and grown overnight 8. The clones on these plates were assayed the following day 209 Screening: Approximately 200 clones of mutants for each position were grown in Zliquid media (384 well microtiter plates) and screened as follows: 1. Overnight cultures in 384-well plates were centrifuged and the media 00 5 removed. To each well was added 0.06 mL 1 mM Tris/S0 4 2 pH 7.8.

2. Made 2 assay plates from each parent growth plate consisting of 0.02 mL cell suspension.

t 3. One assay plate was placed at room temperature and the other at elevated Stemperature (initial screen used 55°C) for a period of time (initially minutes).

4. After the prescribed time 0.08 mL room temperature substrate (TCP saturated 1 mM Tris/S0 4 2 pH 7.8 with 1.5 mM NaN 3 and 0.1 mM bromothymol blue) was added to each well.

Measurements at 620 nm were taken at various time points to generate a progress curve for each well.

6. Data were analyzed and the kinetics of the cells heated to those not heated were compared. Each plate contained 1-2 columns (24 wells) ofunmutated 20F12 controls.

7. Wells that appeared to have improved stability were re-grown and tested under the same conditions.

Following this procedure nine single site mutations appeared to confer increased thermal stability on the enzyme. Sequence analysis was performed to determine of the exact amino acid changes at each position that were specifically responsible for the improvement. In sum, the improvement was conferred at 7 sites by one amino acid change alone, at an eighth site by each of two amino acid changes, and at a ninth site by each of three amino acid changes. Several mutants were then made each having a plurality of these nine beneficial site mutations in combination; of these two mutants proved superior to all the other mutants, including those with single point mutations.

210 Example 6 Direct expression cloning using end-selection An esterase gene was amplified using 5'phosphorylated primers in a standard PCR reaction (10 ng template; PCR conditions: 3' 94 C; 94 C; 1' 50 C; 1'30" 68 0 o C] x 30; 10' 68 C.

O Forward Primer 9511TopF O (CTAGAAGGGAGGAGAATTACATGAAGCGGCTTTTAGCCC) Reverse Primer 951 TopR (AGCTAAGGGTCAAGGCCGCACCCGAGG) The resulting PCR product (ca.1000 bp) was gel purified and quantified.

A vector for expression cloning, pASK3 (Institut fuer Bioanalytik, Goettingen, Germany), was cut with Xba I and Bgl II and dephosphorylated with

CIP.

pmoles Vaccina Topoisomerase I (Invitrogen, Carlsbad, CA) was added to 60 ng (ca. 0.1 pmole) purified PCR product for 5' 37 C in buffer NEB I (New England Biolabs, Beverly, MA) in 5 pl total volume.

The topogated PCR product was cloned into the vector pASK3 (5 pl, ca. 200 ng in NEB I) for 5' at room temperature.

This mixture was dialyzed against H20 for 2 Jtl were used for electroporation of DHIOB cells (Gibco BRL, Gaithersburg, MD).

Efficiency: Based on the actual clone numbers this method can produce 2 x 106 clones per p.g vector. All tested recombinants showed esterase activity after induction with anhydrotetracycline.

211 Example 7 Dehalogenase Thermal Stability This invention provides that a desirable property to be generated by directed evolution is exemplified in a limiting fashion by an improved residual activity (e.g.

00 an enzymatic activity, an immunoreactivity, an antibiotic acivity, etc.) of a molecule

O

c upon subjection to altered environment, including what may be considered a harsh CN enviroment, for a specified time. Such a harsh environment may comprise any Scombination of the following (iteratively or not, and in any order or permutation): an CN 10 elevated temperature (including a temperature that may cause denaturation of a working enzyme), a decreased temperature, an elevated salinity, a decreased salinity, an elevated pH, a decreased pH, an elevated pressure, a decreassed pressure, and an change in exposure to a radiation source (including uv radiation, visible light, as well as the entire electromagnetic spectrum).

The following example shows an application of directed evolution to evolve the ability of an enzyme to regain &/or retain activity upon exposure to an elevated temperature.

Every residue (316) of a dehalogenase enzyme was converted into all 20 amino acids by site directed mutagenesis using 32-fold degenerate oligonucleotide primers.

These mutations were introduced into the already rate-improved variant Dhla 20F 12.

Approximately 200 clones of each position were grown in liquid media (384 well microtiter plates) to be screened. The screening procedure was as follows: 1. Overnight cultures in 384-well plates were centrifuged and the media removed. To each well was added 0.06 mL 1 mM Tris/S0 4 2 pH 7.8.

2. The robot made 2 assay plates from each parent growth plate consisting of 0.02 mL cell suspension.

3. One assay plate was placed at room temperature and the other at elevated temperature (initial screen used 55°C) for a period of time (initially minutes).

212 4. After the prescribed time 0.08 mL room temperature substrate (TCP saturated 1 mM Tris/SO 4 2 pH 7.8 with 1.5 mM NaN 3 and 0.1 mM bromothymol blue) was added to each well. TCP trichloropropane.

Measurements at 620 nm were taken at various time points to generate a progress curve for each well.

0

C

0 6. Data were analyzed and the kinetics of the cells heated to those not heated Swere compared. Each plate contained 1-2 columns (24 wells) ofun-mutated 20F12 controls.

7. Wells that appeared to have improved stability were regrown and tested 10 under the same conditions.

Following this procedure nine single site mutations appeared to confer increased thermal stability on Dhla-20F12. Sequence analysis showed that the following changes were beneficial: D89G F91S TI59L G189Q, G189V I220L N238T W251Y P302A, P302L, P302S, P302K P302R/S306R Only two sites (189 and 302) had more than one substitution. The first 5 on the list were combined (using G189Q) into a single gene (this mutant is referred to as All changes but S306R were incorporated into another variant referred to as Dhla8.

Thermal stability was assessed by incubating the enzyme at the elevated temperature 0 C and 80 0 C) for some period of time and activity assay at 30 0 C. Initial rates 213 V were plotted vs. time at the higher temperature. The enzyme was in 50 mM STris/S0 4 pH 7.8 for both the incubation and the assay. Product (CI) was detected by a standard method using Fe(N0 3 3 and HgSCN. Dhla 20F12 was used as the de facto wild type. The apparent half-life (Ti/ 2 was calculated by fitting the data to an exponential decay function.

00 C< These results are shown in Figure 1.

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