JP2004275194A - Genotype-phenotype mapping molecules and their use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of using a molecule in which a genotype is associated with a phenotype.
SOLUTION: (a) A DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or similar to an amino acid via a spacer at the 3 'end of the obtained RNA. Nucleoside or a substance having a chemical structure skeleton similar to nucleoside, which can be covalently bonded to the substance having the chemical structure skeleton, and (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA. A constructing step of an associating molecule characterized by linking a nucleic acid portion containing a gene and a translation product of the gene, a selecting step of selecting out the associating molecule obtained in the constructing step, and a selecting step. Amplifying the gene portion of the assigning molecule, the method comprising obtaining a DNA encoding a desired protein.
[Selection diagram] FIG.

Description

  The present invention relates to a genotype-phenotype associating molecule, and more particularly, a genotype and phenotype comprising a nucleic acid portion having a nucleotide sequence reflecting the genotype and a protein portion containing a protein involved in the expression of the phenotype. Related molecules. The assigning molecule of the present invention is extremely useful in evolutionary molecular engineering, that is, in the modification of functional biopolymers such as enzymes, antibodies, and ribozymes, and in the creation of biopolymers having functions not found in living organisms. Substance.

  Advances in biochemistry, molecular biology, and biophysics have revealed that organisms are molecular machines that operate and proliferate through the interaction between molecules. The most fundamental property of living things on earth is that genetic information is stored in the nucleotide sequence of DNA, and that information is translated into functional proteins via mRNA. At present, with the development of genetic engineering, biopolymers such as nucleotides and peptides having a given sequence can be easily produced. Today, protein engineering and RNA engineering, which are in the limelight, also benefit from genetic engineering. The goal of protein engineering and RNA engineering is to solve the problem of what the three-dimensional structure of proteins and RNAs that perform specific functions should be, and to allow humans to freely design proteins and RNAs with arbitrary functions. . However, current protein and RNA engineering makes it difficult to theoretically approach the three-dimensional structure due to the diversity and complexity of its structure. Is still at the stage of seeing changes. Therefore, it has not reached the stage of designing proteins and RNA with human wisdom.

  In order to understand the functions of biological macromolecules in conjunction with the elementary processes of higher life phenomena, it is necessary to clarify the structure-function relationships of protein molecules. Our ideas described here borrow not only from "doing the wisdom" but also from "the wisdom of nature". In order to overcome the difficulties of conventional protein engineering and design and produce functional biopolymers as desired by humans, we thought that we had to acquire the ability to make full use of both. By diverting this classical method to design proteins with new functions and activities, it may be possible to avoid the difficulty of protein design due to site-specific mutation. You may say "borrow the wisdom of nature."

The drawback of this method is that it is difficult to screen for mutants with new functions and activities, but RNA catalysis, which has recently been in the limelight, has overcome this difficulty. Attempts have been made to synthesize RNAs of a very large number of random sequences (about 10 ¹³ types) and to select RNAs having specific properties from among them (Non-Patent Document 1).

  This is an example of evolutionary molecular engineering, but as this example symbolically shows, the primary goal of protein evolutionary molecular engineering is to search for a vast sequence space that can never be considered by conventional protein engineering. And select the optimal sequence from them. At this time, if we devised a screening system "exhaustively", we could find many sub-optimal sequences around the optimal sequence, and we could construct an experimental system to study "sequence-function". is there.

  Because the superior functions of living organisms are acquired in the process of evolution, if the evolution can be reproduced, it is possible to modify functional biopolymers such as enzymes, antibodies, ribozymes, etc. in the laboratory, and even find them from living organisms. It should be possible to create biopolymers with no functions. Needless to say, research on protein modification and creation is the most important issue in various aspects of biotechnology such as utilization of enzymes as industrial catalysts, biochips, biosensors, and sugar chain engineering.

  At present, when structural screening is not yet complete, as evolutionary methods are used as more efficient methods, as symbolized by the fact that "screening" is still useful when selecting useful proteins. Worthwhile. If a "time machine" can be created in the laboratory to make evolution more efficient, it will not only modify existing enzymes and proteins such as antibodies (vaccines, monoclonal antibodies), but also decompose enzymes that decompose environmental pollutants It will also open up new ways to create enzymes and new proteins that did not exist in the biological world, such as enzymes and purifying agents. Therefore, if a protein evolution experiment system is established, it can be expected that it can be actively used in many fields such as labor saving and energy saving of industrial processes, energy production, and environmental conservation. The assigning molecule of the present invention is an extremely useful substance in evolutionary molecular engineering such as protein modification.

  Evolutionary molecular engineering refers to the study of molecular design of functional macromolecules by high-speed molecular evolution in a laboratory, that is, to examine the mechanism of adaptive walking in the sequence space of biopolymers in the laboratory and optimize it. This is an area, and is a completely new molecular biotechnology that has begun to produce concrete results in 1990 (Non-Patent Document 2; Non-Patent Document 3).

  Life is the product of molecular evolution and natural selection. Molecular evolution is a universal life phenomenon, but its mechanism is not elucidated only by studies that trace the history of past evolution. Rather, the approach of constructing a simple evolving molecular / biological system in a laboratory and studying its behavior gives basic knowledge on molecular evolution, constructs a verifiable theory, Enable engineering applications.

  It is known that a polymer system will evolve if the following five conditions are satisfied. In other words, (1) an open system far from equilibrium, (2) a self-propagating system, (3) a mutant system, (4) a system that has a genotype-phenotype mapping strategy, and (5) a sequence space A system with appropriate fitness terrain. (1) and (2) are conditions under which natural selection occurs, and (5) is already determined by the physical properties of biopolymers. Evolution by natural selection presupposes the association between genotype and phenotype in (4).

  The following three strategies have been adopted in both nature and evolutionary molecular engineering. That is, (a) a ribozyme type that puts a gene and a phenotype on the same molecule, (b) a virus type that forms a complex of a genotype and a phenotype, and (c) a genotype and a phenotype are put in one bag. Cell type (FIG. 1).

  The ribozyme type (a), in which the genotype and phenotype are put on the same molecule, is a simple system and has been successfully used with an RNA catalyst (ribozyme) (Non-patent Document 4).

  Problems of the cell type (c) include (1) averaging effect, (2) bizarre effect, and (3) random replication effect. The averaging effect occurs when the number of copies of the genome of the cell is large, because the association between the genotype and the phenotype is statistically averaged and becomes ambiguous. In cells, performance improvement is averaged because it is only one of the copy numbers (n), and competition for survival in the cell population starts at a selection coefficient (s) / n. Therefore, it is advantageous for the cell type that the copy number (n) is as small as possible. However, when the number of segments is large due to the bizarre effect, n must be very large to prevent the bizarre effect. Therefore, the apparent selection coefficient in the survival competition in the cell population is expected to be extremely small as compared with the virus type. Since the time required for selection is proportional to the reciprocal of the selection coefficient, the evolution rate is much slower than that of the virus type. Furthermore, the random replication effect of (3) is lethal for cell types. This effect is due to the fact that it is extremely difficult to replicate all of the essential genes before cell division, because the segmented essential genes are replicated randomly. This means that, even if a beneficial mutation occurs in an essential gene, the probability that it will be replicated and transmitted to daughter cells is extremely small.

  In order to evolve efficiently, it is necessary to integrate the genotype and the phenotype as in the virus type of (b).

  Phage display (Non-patent document 5; Non-patent document 6), polysome display (Non-patent document 7), and code have already been used as evolutionary molecular engineering of a virus type that forms a genotype-phenotype complex of (b). Various methods have been proposed and are being developed, including a library with a generalized tag (Non-Patent Document 8) and a cell stat (Non-Patent Document 9).

  However, despite the importance of the sequence space that can be explored is important in evolutionary molecular engineering, a global exploration method for the sequence space similar to the ribozyme type has not yet been established for these virus types.

  The reason for this is that when a current virus such as phage display is used, it is inevitably subject to the following limitations depending on the host cell because it is parasitic on the current cell. In other words, (1) it is restricted by cells, so that only limited sequence space can be probed, (2) membrane permeability, (3) bias by host, (4) library limitation by host population, etc. is there.

  The polysome display method (Patent Document 1) is non-covalently linked to a nucleic acid and a protein via a ribosome, and thus is suitable for a peptide having a short chain length, but has a chain length like a protein. If it gets longer, its handling becomes a problem. In particular, since a huge ribosome is kept added, conditions for selection (for example, adsorption and elution) are strongly restricted by conditions. The library with a coded tag (Patent Document 2) associates a nucleic acid tag with a peptide chemically synthesized via beads, but with current technology, chemical synthesis of a protein of about 100 residues has a very high yield. Can be used for peptides with short chains, but not for proteins with long chains.

One way to overcome these problems is to use a cell-free translation system. The advantages of a viral-type strategic molecule that simply combines genotype and phenotype in a cell-free system are as follows: (1) it can synthesize an enormous population of mutants that can be ribozyme-type, and (2) it depends on the host. (3) there is no problem of membrane permeability, (4) the 21st code can be used and unnatural amino acids can be introduced, and so on.
Ellington, AD & Szostak, JW, 1990, Nature, 346, p.818-822 Joe Fushimi, 1991, Science, Vol.61, p.333-340 Joe Fushimi, "Lecture Evolution (Vol.6)", The University of Tokyo Press, 1992 Hiroshi Yanagawa, "New Age of RNA", Yodosha, 1993, p.57-77 Smith, GP, 1985, Science, 228, p.1315-1317 Scott, JK & Smith, GP, 1990, Science, 249, p.386-390 Mattheakis, LC et al., 1994, Proc. Natl. Acad. Sci. USA, 91, p. 9022-9026 Brenner, S. & Lerner, RA, 1992, Proc. Natl. Acad. Sci. USA, 89, p. 5381-5383. Husimi, Y. et al., 1982, Rev. Sci. Instrum. 53, p.517-522 WO 95/11922 pamphlet WO 96/22391 pamphlet

  An object of the present invention is to provide a virus-type working replicon that has the advantages of the above-mentioned virus-type strategic molecule, is more efficient than phage, and has less restrictions on the setting of environmental conditions, that is, between nucleic acids and proteins, which should be called in vitro viruses. It is an object of the present invention to provide a molecule linked by a chemical bond, that is, a molecule in which a genotype is associated with a phenotype. More specifically, the present invention uses a cell-free protein synthesis system to associate a genotype (nucleic acid) with a phenotype (protein), which can be used for creation of a functional protein or peptide, and to perform a The purpose of the present invention is to provide a molecule having a 1: 1 correspondence between information and function by connecting the 3 'end of the gene and the C end of the protein by a covalent bond. In addition, the formed genotype-phenotype mapping molecule (hereinafter, also referred to as “in vitro virus”) is selected by an in vitro selection method, and the gene portion of the selected in vitro virus is amplified by reverse transcription PCR. It is another object of the present invention to search an enormous sequence space and obtain a target functional protein or peptide by repeating an operation of amplifying while introducing a mutation.

  The present inventors have conducted intensive studies to achieve the above object, and as a result, two types of genotype and phenotype associating molecules in which nucleic acids and proteins are chemically bonded on the ribosome of a cell-free protein synthesis system can be constructed. I found that. Further, the assigning molecule (in vitro virus) is selected by an in vitro selection method, the gene portion of the selected in vitro virus is amplified by reverse transcription PCR, and further, a protein evolution experiment system for amplifying while introducing mutations. Found that it could be built. The present invention has been accomplished based on these findings.

  That is, the present invention includes a nucleic acid portion having a nucleotide sequence reflecting a genotype, and a protein portion containing a protein involved in phenotypic expression, wherein the nucleic acid portion and the protein portion are directly chemically bonded. And phenotype mapping molecules.

  According to a preferred embodiment of the present invention, the assigning molecule described above, wherein the 3 ′ end of the nucleic acid portion and the C terminal of the protein portion are covalently bonded, and the 3 ′ of the nucleic acid portion covalently bonded to the C terminal of the protein portion. An assigning molecule as described above, wherein the terminus is puromycin, is provided.

  Further, according to a preferred aspect of the present invention, there is provided the above-mentioned assigning molecule, wherein the nucleic acid part contains a gene encoding a protein, and the protein part is a translation product of the gene of the nucleic acid part. The nucleic acid portion preferably contains a gene consisting of RNA and a suppressor tRNA linked to the gene via a spacer. The suppressor tRNA preferably contains an anti-codon corresponding to the stop codon of the gene. Alternatively, the nucleic acid part includes a gene composed of RNA and a spacer part composed of DNA and RNA or DNA and polyethylene glycol. Further, the nucleic acid part may include a gene composed of DNA and a spacer part composed of DNA and RNA.

Further, according to another embodiment of the present invention, (a) a DNA having a sequence corresponding to the suppressor tRNA is ligated to the 3 ′ end of DNA containing the gene via a spacer, and (b) the obtained DNA ligated product is transcribed. (C) connecting a substance having a nucleoside or a nucleoside-like chemical structural skeleton capable of covalently bonding to an amino acid or a substance having a chemical structural skeleton similar to an amino acid to the 3 ′ end of the obtained RNA; (D) associating a genotype with a phenotype, wherein protein synthesis is performed in a cell-free protein synthesis system using the obtained conjugate as mRNA, and a nucleic acid part containing the gene is linked to a translation product of the gene. The method of constructing the molecule, (a) preparing DNA containing a gene without a stop codon, (b) transcribing the prepared DNA into RNA, and (c) placing DNA on the 3 ′ end side of the obtained RNA. And RNA chimera spacer (D) further linking a substance having a nucleoside or a nucleoside-like chemical structural skeleton, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, to the 3′-terminal side of the obtained conjugate; (E) associating a genotype with a phenotype, wherein protein synthesis is performed in a cell-free protein synthesis system using the obtained conjugate as mRNA, and a nucleic acid part containing the gene is linked to a translation product of the gene. Methods for constructing molecules are provided.

  Further, according to a preferred aspect of the present invention, there is provided the above-mentioned construction method, wherein the nucleoside or the substance having a chemical structure skeleton similar to the nucleoside is puromycin.

  According to another embodiment of the present invention, (a) DNA containing a gene without a stop codon is prepared, (b) the prepared DNA is transcribed into RNA, and (c) 3 ′ of the obtained RNA. A nucleoside capable of linking a chimeric spacer of DNA and polyethylene glycol to the terminal side and (d) further covalently bonding to the 3 ′ terminal side of the obtained conjugate a amino acid or a substance having a chemical structure skeleton similar to an amino acid; Alternatively, a substance having a chemical structural skeleton similar to a nucleoside is linked, and (e) protein synthesis is performed in a cell-free protein synthesis system using the obtained link as mRNA, and a nucleic acid portion containing a gene is linked to a translation product of the gene. And a method for constructing a genotype-phenotype associating molecule, characterized in that

  According to another embodiment of the present invention, (a) a DNA containing a gene without a stop codon is prepared, (b) the prepared DNA is transcribed into RNA, and (c) 3 ′ of the obtained RNA. A double-stranded DNA spacer is linked to the terminal side, and (d) a nucleoside or a nucleoside which can be covalently bonded to an amino acid or a substance having a chemical structure skeleton similar to the amino acid on the 3 ′ end side of the obtained conjugate. A substance having a chemical structural skeleton similar to a nucleoside is ligated, and (e) the obtained conjugate is used as mRNA to perform protein synthesis in a cell-free protein synthesis system, and a nucleic acid portion containing a gene is ligated to a translation product of the gene. And a method for constructing a genotype-phenotype associating molecule.

  Furthermore, according to another embodiment of the present invention, (a) a DNA containing a gene having no stop codon and a base sequence of a spacer is prepared, (b) the prepared DNA is transcribed into RNA, and A substance having a nucleoside or a nucleoside-like chemical structural skeleton, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, was linked to the 3′-terminal side of the obtained RNA to obtain (d). Short-chain PNA or DNA is added to the 3'-terminal portion of the gene portion of the RNA conjugate to form a double strand, and (e) the resulting conjugate is used as mRNA for protein synthesis in a cell-free protein synthesis system. And linking a nucleic acid portion containing the gene with a translation product of the gene, thereby providing a method for constructing a genotype-phenotype associating molecule.

  According to still another aspect of the present invention, a construction step of constructing an associating molecule from DNA containing a gene by the above-described construction method, a culling step of culling out the associating molecule obtained in the construction step, A method for experimentally evolving a protein, comprising: a mutation introduction step of introducing a mutation into the gene portion of the assigning molecule selected in the step; and an amplification step of amplifying the gene portion obtained in the mutation introduction step. Is done.

In the evolution experiment method, preferably, the construction step, the selection step, the mutation introduction step, and the amplification step are repeatedly performed by subjecting the DNA obtained in the amplification step to the construction step. Further, a first ligation means for ligating the DNA of the sequence corresponding to the suppressor tRNA to the 3 'end of the DNA containing the gene via a spacer, a transcription means for transcribing the DNA ligated product obtained by the first ligation means to RNA ,
Second linking means for linking a substance having a nucleoside or a chemical structure skeleton similar to a nucleoside, which can be covalently bonded to an amino acid or a substance having a chemical structure skeleton similar to an amino acid, to the 3 ′ end of the RNA obtained by the transcription means And an associating molecule comprising a ligated product obtained by the second ligating means as mRNA, protein synthesis in a cell-free protein synthesis system, and a third ligating means for linking a nucleic acid portion containing the gene and a translation product of the gene. Constructing means, or a transcription means for transcribing DNA containing a gene to RNA, a chimera of DNA and RNA or a chimera of DNA and polyethylene glycol or a chimera of DNA and DNA at the 3 'end of RNA obtained by the transcription means. First connecting means for connecting a double-stranded spacer consisting of a single-stranded or RNA and a short-chain peptide nucleic acid (PNA) or DNA, and an RNA-space obtained by the first connecting means A second linking means for linking a substance having a nucleoside or a chemical structure skeleton similar to a nucleoside, which can be covalently bonded to an amino acid or a substance having a chemical structure skeleton similar to an amino acid, Performing protein synthesis in a cell-free protein synthesis system using the ligated product obtained by the two ligating device as mRNA, a nucleic acid portion containing a gene, and an associating molecule constructing device including a third ligating device for linking a translation product of the gene, A means for selecting the constructed mapping molecule, a means for introducing a mutation into the gene portion of the selected mapping molecule, and a means for amplifying the mutated gene portion, An apparatus for performing the evolution experiment method of is also provided.

  Further, according to another aspect of the present invention, a construction step of constructing an assigning molecule by the above construction method, and an assay step of examining the interaction between the assigning molecule obtained in the construction step and another protein or nucleic acid, A method for assaying protein-protein or protein-nucleic acid interaction is provided.

More specifically, the following is provided.
(1) (a) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or amino acid similar to the amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having a chemical structural skeleton, and (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, A step of constructing an associating molecule characterized by linking a nucleic acid portion containing the gene and a translation product of the gene, and a step of selecting out the associating molecule obtained in the constructing step. A method for selecting a desired protein.
(2) (a) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or amino acid similar to the amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having a chemical structural skeleton, and (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, A step of constructing an assigning molecule characterized by linking a nucleic acid portion containing the gene and a translation product of the gene, a step of selecting the assigning molecule obtained in the constructing step, and a step of selecting An amplification step of amplifying the gene portion of the assigning molecule, and by subjecting the DNA obtained in the amplification step to a construction step, the construction step, the selection step, and the amplification step are repeatedly performed. Selection method of the desired protein.
(3) (a) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or amino acid-like amino acid is obtained via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having a chemical structural skeleton, and (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, A step of constructing an assigning molecule characterized by linking a nucleic acid portion containing the gene and a translation product of the gene, a step of selecting the assigning molecule obtained in the constructing step, and a step of selecting An amplification step of amplifying a gene portion of the assigning molecule. A method for obtaining a DNA encoding a desired protein.
(4) The method for obtaining DNA according to (3), wherein the DNA obtained in the amplification step is subjected to a construction step, whereby the construction step, the selection step, and the amplification step are repeated.
(5) (a) preparing DNA containing a gene, (b) transcribing the prepared DNA into RNA, (c)
A substance having a nucleoside or a nucleoside-like chemical structural skeleton, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end side of the obtained RNA, is linked (d A) constructing an associating molecule, comprising performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA and linking a nucleic acid portion containing the gene and a translation product of the gene; A method for selecting RNA encoding a desired protein, comprising:
(6) (a) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or amino acid similar to the amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having a chemical structural skeleton, and (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, A step of constructing an assigning molecule characterized by linking a nucleic acid portion containing the gene and a translation product of the gene, a step of selecting the assigning molecule obtained in the constructing step, and a step of selecting An amplification step of amplifying the gene portion of the assigning molecule, and by subjecting the DNA obtained in the amplification step to a construction step, the construction step, the selection step, and the amplification step are repeatedly performed. RNA selection method of encoding the desired protein.
(7) The method according to any one of (1) to (6), wherein the DNA containing the gene further contains an expression control region.
(8) The method according to any one of 1 to 7, wherein the spacer comprises a polymer substance.
(9) The method according to any one of (1) to (8), wherein the length of the spacer is in the range of 100 to 1000 °.
(10) The method according to any one of (1) to (9), wherein the spacer contains a nucleic acid.
(11) the nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or 11. The method according to 10, which is a double strand of a single strand consisting of RNA and DNA and a short strand DNA.
(12) The method according to any one of (1) to (11), wherein the spacer contains polyethylene glycol.
(13) The method according to 12, wherein the polyethylene glycol has a molecular weight of 3,000 to 30,000.
(14) The method according to 9 or 13, wherein the spacer comprises polyethylene glycol and DNA.
(15) The method according to any one of 1 to 14, wherein the nucleoside or the substance having a chemical structural skeleton similar to the nucleoside is puromycin or a derivative thereof.
(16) The method according to any one of (1) to (15), wherein the link between the nucleic acid portion containing the gene and the translation product of the gene is formed by a covalent bond.
(17) The method according to 16, wherein the covalent bond is an amide bond.
(18) The method according to any one of (1) to (17), wherein the assigning molecule selected in the selection step is selected using an interaction with a substance as an index.
(19) The method according to 18, wherein the selection step includes a step of separating a complex by an interaction between the substance bound to the solid phase and the assigning molecule obtained in the construction step.
(20) The method according to any one of 1 to 19, wherein the gene is an antibody or a partial gene thereof.
(21) The method according to any one of 1 to 19, wherein the gene is an enzyme or a partial gene thereof.
(22) The method according to any one of (1) to (21), wherein the DNA containing the gene is a library comprising a plurality of DNAs having different genes.
(23) The method according to any one of 1 to 22, wherein the DNA containing the gene comprises a random base sequence.

(24) A molecule in which a spacer made of a high molecular substance is bonded to the 3 ′ end of RNA having a gene.
(25) A molecule having a nucleoside or a substance having a chemical structural skeleton similar to a nucleoside, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, to the 3 ′ terminal side of the molecule according to 24.
(26) The molecule according to 24 or 25, wherein the RNA further comprises an expression control region.
(27) The molecule according to any one of (24) to (26), wherein the length of the spacer is in the range of 100 to 1000 °.
(28) The molecule according to any of 24-27, wherein the spacer comprises a nucleic acid.
(29) the nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or 29. The molecule according to 28, which is a double-stranded single-stranded DNA comprising RNA and DNA and a short-stranded DNA.
(30) The molecule according to any one of 24-29, wherein the spacer comprises polyethylene glycol.
(31) The molecule according to 30, wherein the spacer comprises polyethylene glycol and DNA.
(32) The method according to 30 or 31, wherein the molecular weight of the polyethylene glycol is 3,000 to 30,000.
(33) The molecule according to any of 25 to 32, wherein the nucleoside or the substance having a chemical structure skeleton similar to the nucleoside is puromycin or a derivative thereof.
(34) The molecule according to any of 24-33, wherein the gene is an antibody or a part of the gene.
(35) The molecule according to any of 24-33, wherein the gene is an enzyme or a partial gene thereof.
(36) The molecule according to any of 24-33, wherein the gene comprises a random base sequence.
(37) The library according to any one of (24) to (35), comprising a plurality of molecules having different genes.

(38) A molecule in which a nucleic acid portion having a nucleotide sequence reflecting a genotype is directly covalently bonded to a protein portion containing a protein encoded by the nucleotide sequence reflecting the genotype, wherein the nucleic acid portion is a DNA. Including molecules.
(39) The molecule according to 38, wherein the nucleic acid portion further contains an expression control region.
(40) The molecule according to (38) or (39), wherein the DNA contained in the nucleic acid portion is a double strand of DNA and RNA.
(41) The molecule according to 38 or 39, wherein the DNA contained in the nucleic acid portion is a single strand of DNA.
(42) The molecule according to 38 or 39, wherein the DNA contained in the nucleic acid portion is a double-stranded DNA.
(43) The molecule according to any of (38) to (42), wherein the DNA contained in the nucleic acid portion is generated by reverse transcription of RNA.
(44) A nucleic acid moiety similar to a nucleoside or a nucleoside capable of covalently bonding to a substance having a chemical structural skeleton similar to an amino acid or an amino acid via a spacer on the 3′-terminal side of the nucleotide sequence reflecting the genotype, 44. The molecule according to any of 38 to 43, which is a molecule to which a substance having a chemical structural skeleton is bonded.
(45) The molecule according to 44, wherein the nucleoside or the substance having a chemical structural skeleton similar to the nucleoside is puromycin or a derivative thereof.
(46) The molecule according to (44) or (45), wherein the spacer comprises a polymer substance.
(47) The molecule according to any one of (44) to (46), wherein the spacer has a length of 100 to 1000 °.
(48) The molecule according to any one of 44 to 47, wherein the spacer comprises a nucleic acid.
(49) the nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or 49. The molecule according to 48, wherein the molecule is a double strand of a single strand consisting of RNA and DNA and a short strand of DNA.
(50) The molecule according to any of 44 to 49, wherein the spacer comprises polyethylene glycol.
(51) The molecule according to 50, wherein the spacer comprises polyethylene glycol and DNA.
(52) The method according to 50 or 51, wherein the polyethylene glycol has a molecular weight of 3,000 to 30,000.
(53) The molecule according to any one of 38 to 52, wherein the gene is an antibody or a partial gene thereof.
(54) The molecule according to any one of 38 to 52, wherein the gene is an enzyme or a partial gene thereof.
(55) The molecule according to any one of 38 to 52, wherein the gene comprises a random base sequence.
(56) The library according to any one of (38) to (54), comprising a plurality of molecules having different genes.

  According to the present invention, an associating molecule between a genotype (nucleic acid part) and a phenotype (protein part) and a method for constructing the same are provided. In addition, the assigning molecule (in vitro virus) constructed according to the present invention is selected by an in vitro selection method, a very small amount of the selected in vitro virus gene portion is amplified by reverse transcription PCR, and a mutation is further introduced. The present invention also provides a method for experimentally evolving a protein using an in vitro virus, which is characterized in that the amplification is performed while the protein is amplified. The genotype and phenotype mapping molecule of the present invention and the method of experimenting the evolution of a protein using the same are based on evolutionary molecular engineering, that is, modifying functional biopolymers such as enzymes, antibodies, and ribozymes. It is an extremely useful substance or experimental system that can be used in the creation of biopolymers with functions not found in living organisms.

  In this specification, a number of terms are used, and as used herein, those terms have the following meanings. "Nucleic acid part" refers to a substance having a nucleoside or a chemical structure skeleton similar to a nucleoside such as RNA, DNA, PNA (Peptide nucleic acid, peptide nucleic acid; a polymer in which nucleobases are linked via an amino acid analog), and the like. And the “protein part” means a conjugate of a substance having an amino acid such as a natural amino acid or an unnatural amino acid or a chemical structural skeleton similar to an amino acid. The term “suppressor tRNA” refers to a tRNA that can suppress mutation by changing its structure, such as reading a stop codon on mRNA as a codon corresponding to a certain amino acid. “Having a nucleotide sequence that reflects the genotype” means that a gene or a portion thereof related to the genotype is included. "Includes proteins involved in phenotypic expression" means that proteins that express themselves become phenotypic traits or that are involved in the expression of phenotypic traits by acting as enzymes, etc. I do.

  The spacer located on the 3 'side of the nucleic acid portion may be any spacer as long as it is preferably a polymer substance having a length of 100 mm or more, more preferably about 100 to 1000 mm. Specifically, a single strand of natural or synthetic DNA or RNA, a double strand of DNA and DNA, a double strand of RNA and a short strand (for example, about 15 to 25 nucleotides) of PNA or DNA, a polysaccharide, etc. And organic synthetic high molecular substances such as polyethylene glycol, preferably polyethylene glycol having a molecular weight of about 3,000 to 30,000.

  The nucleic acid portion and the protein portion of the assigning molecule of the present invention are linked by a chemical bond such as a covalent bond. In particular, a substance having a chemical structure skeleton similar to a nucleoside or a nucleoside at the 3 ′ end of a nucleic acid part or a continuum thereof, and a substance having a chemical structure skeleton similar to an amino acid or an amino acid at the C terminal of the protein part are chemically bonded, for example. Those covalently bonded are preferred.

  For binding of the nucleic acid part and the protein part, for example, puromycin (Puromycin) having an amide bond as a chemical bond at the 3 ′ end of the nucleic acid part, 3′-N-aminoacylpuromycin aminonucleoside (3′-N-Aminoacylpuromycin aminonucleoside) , PANS-amino acids), for example, PANS-Gly of glycine, PANS-Val of valine, PANS-Ala of alanine, and PANS-all amino acids corresponding to all amino acids. In addition, 3'-N-aminoacyladenosine aminonucleoside (3'-Aminoacyladenosine aminonucleoside, AANS-amino acid For example, AANS-Gly of glycine, AANS-Val of valine, AANS-Ala of alanine, and other AANS-all amino acids corresponding to all amino acids can be used. Further, nucleosides or those in which a nucleoside and an amino acid are ester-bonded can also be used. In addition, any of nucleosides or substances having a chemical structural skeleton similar to nucleosides and amino acids or substances having a chemical structural skeleton similar to amino acids can be used as long as they can be chemically bonded.

  The genotype-phenotype assigning molecules of the present invention include, for example, the following methods (1) in which the binding between the nucleic acid portion and the protein portion is site-specific, or (2) when the binding between the nucleic acid portion and the protein portion is non-site. Can be constructed in a specific way.

  First, (1) a method in which the binding between the nucleic acid part and the protein part is site-specific will be described.

  In this method, (a) a DNA having a sequence corresponding to the sup tRNA is ligated to the 3 ′ end of the DNA containing the gene via a spacer, and (b) the obtained DNA conjugate is transcribed into RNA, (C) A substance having a nucleoside or a nucleoside-like chemical structural skeleton, such as puromycin, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, is linked to the 3 ′ end of the obtained RNA. (D) using the obtained conjugate as mRNA to perform protein synthesis in a cell-free protein synthesis system, for example, a cell-free protein synthesis system of Escherichia coli, thereby obtaining (e) a gene RNA (genotype) and its translation product protein. (Phenotype) refers to the phenotype of a genotype chemically linked via a nucleoside or a substance having a chemical structural skeleton similar to a nucleoside, for example, puromycin. It is possible to construct a response with molecules.

  That is, according to the method of the present invention, in the protein synthesis, when a stop codon comes to the A site of the ribosome, the sup tRNA enters correspondingly, and by the action of peptidyl transferase, the nucleoside at the 3 ′ end of the sup tRNA or Substances having a chemical structure skeleton similar to nucleosides, such as puromycin, bind to proteins (FIG. 2). Therefore, this method is site-specific in that the binding between the nucleic acid part and the protein depends on the genetic code.

  Puromycin (FIG. 3) is a bacterium (Nathans, D. (1964) Proc. Natl. Acad. Sci. USA, 51, 585-592; Takeda, Y. et al. (1960) J. Biochem. 48, 169). -177) and proteins of animal cells (Ferguson, JJ (1962) Biochim. Biophys. Acta 57, 616-617; Nemeth, AM & de la Haba, GL (1962) J. Biol. Chem. 237, 1190-1193) It is known to inhibit synthesis. The structure of puromycin is similar to the structure of aminoacyl-tRNA, which reacts with the peptidyl-tRNA bound to the P-site of the ribosome and is released from the ribosome as peptidyl-puromycin, interrupting protein synthesis (Harris, RJ ( 1971) Biochim. Biophys. Acta 240, 244-262).

The method of purifying a native sup tRNA and linking it to mRNA is not practical because of problems such as purification of the sup tRNA and easiness of hydrolysis of an ester bond between the amino acid and the 3 ′ end of the tRNA. So far, studies on tRNA identity have shown that unmodified tRNA is aminoacylated in the same way as intact tRNA, and that aminoacylated unmodified tRNA is incorporated into ribosomes and translated. (Shimizu, M. et al. (1992) J. Mol. Evol. 35, 436-443). Also, the identity of the tRNA has been used to create the sup tRNA.

  It is known that aminoacyl synthases of alanine, histidine and leucine do not recognize these anticodons (Tamura, K. et al. (1991) J. Mol. Recog. 4, 129-132). Therefore, when the anticodon of the alanine tRNA is changed to a stop codon (for example, amber), it can be expected that the alanine tRNA (sup tRNA) will be incorporated corresponding to the stop codon.

The problem here is whether, unlike normal tRNA, tRNA whose 5 ′ side is not shaped by RNAse P or the like enters the ribosome A site. This is the most important issue that needs to be investigated when deciding whether this model is acceptable. Brome Mosaic Virus (BMV) and Turnip Yellow Mosaic Virus (TYMV) have a 3'-terminal tRNA-like structure, are aminoacylated by aminoacyl synthetase, and incorporate amino acids at a 1% efficiency in a cell-free translation system. (Chen, JM & Hall, TC (1973) Biochemistry 12,
4570-4574). If even 1% of the BMV RNA is incorporated into the ribosome, RNA with an intact tRNA at the 3 'end may enter the RNA a little more efficiently. Even if the intact tRNA is taken in with an efficiency of 10% or less, the concentration effect may be sufficient to compete with the release factor.

  Therefore, before the experiment of binding the protein to the 3 'end of mRNA-sup tRNA (the one in which the sup tRNA was linked to the 3' side of the mRNA via a spacer), even the sup tRNA separated from the mRNA was placed on the A site of the ribosome. I went into it to see if it could bind to proteins. Actually, a sup tRNA in which puromycin is bound to the 3 ′ end of the sup tRNA is prepared, and the sup tRNA is introduced into a cell-free protein synthesis system.The sup tRNA portion enters the stop codon of the A site of the ribosome, It was examined whether it binds to proteins. The mRNA used a four repeat region (127 residues) of tau protein (Goedert, M. (1989) EMBO J. 8, 392-399). As a result, it was confirmed that the sup tRNA having puromycin at the 3 'end was inserted corresponding to the termination codon of the A site of the ribosome and was bound to the protein by translation in a cell-free protein synthesis system (FIG. 2). ).

  Next, construct an RNA-sup tRNA conjugate in which the length of the spacer between the mRNA and the sup tRNA is changed, and adjust the optimal length of the spacer in which the sup tRNA portion is most efficiently incorporated into the A site of the ribosome. We tried to select by in vitro selection method. As a result, it was found that the RNA-sup tRNA conjugate having a certain spacer length efficiently and chemically binds to the protein of the translation product.

  In order to construct the genotype-phenotype mapping molecule of the present invention, first, a nucleoside or nucleoside that can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid attached to the 3 ′ end of a nucleic acid portion is used. It is necessary to synthesize a substance having a chemical structural skeleton similar to that of, for example, 2′-deoxycytidylyl puromycin (dCpPur) or ribocytidyl puromycin (rCpPur) (FIG. 3).

An example of a method for synthesizing dCpPur is as follows. First, puromycin-5'monophosphate can be produced by chemically phosphorylating the 5 'hydroxyl group of puromycin with phosphorus oxychloride and trimethyl phosphate. Next, the amino group of the amino acid portion of the puromycin-5'monophosphate and the 2'hydroxyl group of the ribose portion can be protected by reacting puromycin-5'monophosphate with trifluoroacetic acid and trifluoroacetic anhydride. To this, after reacting Bz-DMT deoxycytidine, which protected the amino group in the pyrimidine ring of deoxycytidine and the 5 ′ hydroxyl group of the ribose moiety, in the presence of a condensing agent, dicyclohexylcarbodiimide, deprotection was performed with acetic acid and ammonia. , 2'-deoxycytidylyl puromycin (dCpPur). By phosphorylating the 5 ′ hydroxyl group of dCpPur with polynucleotide kinase, pdCpPur can be obtained.

  Ribocytidyl puromycin can be prepared by condensing puromycin with a protected rC-beta amidite in the presence of tetrazole, followed by oxidation and deprotection.

  Next, the construction of a conjugate constituting the nucleic acid portion for connecting the nucleic acid portion and the protein portion in a site-specific manner will be described.

  Examples of the conjugate constituting the nucleic acid portion used in the site-specific method include a 5'-T7 promoter region-Shine-Dalgarno sequence (SD) region-mRNA region-spacer region-sup tRNA region-puromycin region-3. 'Can be mentioned.

  In constructing the linked nucleic acid portion, first, a four repeat region, which is a microtubule binding region of a human tau protein called htau24 (Goedert, M. (1989) EMBO J. 8, 392-399), is inserted into the T7 promoter. A plasmid (pAR3040) inserted downstream is constructed, and cut with restriction enzymes BglII and BamHI to obtain a linear DNA. Using this DNA as a template, PCR is performed with Taq DNA polymerase using primers on the upstream side (forward) including the T7 region and on the downstream side (backward) including the SD region and the region near the start codon, to amplify.

  At this time, in the backward primer, three methionines are added in order to increase the detection sensitivity of radioactive methionine in the protein part after protein synthesis. That is, the 4th leucine and the 5th and 8th lysines in the 4 repeat region are respectively substituted with methionine. Ultimately, the translated four repeat protein contains a total of four methionines. Next, using the above-mentioned backward primer and the complementary strand as the forward primer, and using the primer designed so that the amber codon comes as a stop codon at the C-terminus of the 4 repeats, the backward primer was used. Amplification is performed by PCR using the DNA containing the region as a template.

  The two DNA fragments amplified by PCR, that is, the DNA fragment containing the T7 promoter and the SD region and the DNA fragment containing the four repeat regions are mixed together, first extended without a primer, and then the primer containing the sequence of the T7 promoter is forwarded. Using a primer containing a stop codon at the C-terminus of the 4-repeat region as the backward, amplify again by PCR.

  A double-stranded DNA fragment consisting of 17 residues having cohesive ends at both ends is tandemly ligated to this DNA ligated product (T7 promoter-SD-4 repeat) with DNA ligase to form ligated products having different spacer lengths. .

  After ligation, the mixture is fractionated into three fractions (a, b, c) based on the length by polyacrylamide electrophoresis (PAGE). The spacer is (17) n, the fraction a is n = 15-18, the fraction b is n = 6-14, and the fraction c is n = 0-5. The sup tRNA is prepared by chemically synthesizing a sequence of a natural alanine tRNA at several positions and an anticodon sequence modified to amber (UAG). Conjugates of a, b, and c fractions having different spacer lengths are ligated to this sup tRNA using T4 DNA ligase. An excess amount of single-stranded backing DNA is used at the joining site, and the temperature is once raised and melted, then annealed to form a complementary strand, and then joined. After ligation, amplification is performed by PCR using primers at the 5 'end and 3' end of the conjugate. This DNA conjugate is transcribed using T7 RNA polymerase to form an RNA conjugate.

By linking the previously chemically synthesized pdCpPur to the 3 'end of this RNA conjugate with T4 RNA ligase, an RNA conjugate that can be used as a cell-free protein synthesis system gene, 5'-T7 promoter region-SD Region-4 repeat region-spacer region-sup tRNA region-puromycin-3 'can be obtained.

  The above-described RNA conjugate is added as mRNA to a cell-free protein synthesis system, for example, a cell-free protein synthesis extract such as Escherichia coli or rabbit reticulocyte, to perform protein synthesis. The following experiment is performed to determine the optimal spacer length for the most efficient connection between the nucleic acid part (RNA) and the protein part.

  That is, protein synthesis is performed using a cell-free protein synthesis system using the above-described RNA conjugate having three different spacer lengths, a, b, and c fractions as genes. At this time, when a tRNA charged with modified lysine in which lysine is linked to biotin via its ε-amino group is added, biotinyl lysine is incorporated into some translated lysine residues of the 4-repeat protein. After protein synthesis, magnetic beads with streptavidin linked to the surface are added, and the biotin-loaded protein is caught.

  If the protein part is linked to the nucleic acid part (RNA) via puromycin, the nucleic acid part (RNA) should be attached to the C-terminal of the protein. Reverse transcription was performed using the sequence corresponding to the N-terminal region of 4 repeats as the forward primer and the 3 'end of the sup tRNA as the backward primer to confirm whether the RNA-protein conjugate was really caught by the magnetic beads. After performing polyacrylamide gel electrophoresis, DNA bands reverse-transcribed only for the spacer length of the c fraction are confirmed. This means that the RNA conjugate having the spacer length of the c fraction is most efficiently linked to the protein part.

  Next, (2) a method in which the binding between the nucleic acid part and the protein part is non-site-specific will be described.

  In this method, (a) DNA containing a gene without a stop codon is prepared, (b) the prepared DNA is transcribed into RNA, and (c) DNA is added to the 3 ′ end of the obtained RNA. A substance having a nucleoside or a nucleoside-like chemical structural skeleton capable of covalently bonding to an amino acid or a substance having a chemical structural skeleton similar to an amino acid at the 3'-terminal thereof by linking a chimeric RNA spacer. For example, puromycin is ligated, and (e) the resulting conjugate is used as mRNA to perform protein synthesis in the above cell-free protein synthesis system, for example, a cell-free protein synthesis system of Escherichia coli. A genotype-phenotype associating molecule can be constructed in which the protein of the translation product is chemically linked via puromycin.

  That is, according to the method of the present invention, a substance having a chemical structure skeleton similar to a nucleoside or a nucleoside at the 3 ′ end of the nucleic acid portion, for example, puromycin is located at the A site of the ribosome, corresponding to the termination codon of mRNA on the ribosome. Instead, the puromycin at the 3 'end of the RNA-DNA chimeric nucleic acid is chemically bound to the protein by the action of the peptidyl transferase (Fig. 4). Therefore, this method is site-specific, in which the binding between the nucleic acid portion and the protein is independent of the genetic code.

  In this method, a genotype and phenotype associating molecule can be constructed in the same manner as in the site-specific method described in (1) above, using a site-specific one for the linked body of nucleic acid portions.

  As a conjugate of the nucleic acid portion used in the site-unspecified method, for example, 5'-T7 promoter region-Shine-Dalgarno sequence (SD) region-mRNA region-spacer region-puromycin region-3 'were connected in this order. A linked body can be mentioned.

  In the construction of the conjugate of the nucleic acid part, first, the construction of the conjugate from the T7 promoter region to the end of the four-repeat translation region is carried out in the above (1) Construction of the conjugate of the nucleic acid part by the site-specific method. According to the method described above, the difference is that, when amplifying by PCR using the ligation construct constructed as described above as a template, two C-terminal stop codons of four repeats, ocher (CTG) and amber (TAA) were added to the backward primer. ) To CAG (glutamine) and AAA (lysine), respectively, and using primers designed to eliminate the stop codon.

  Using the DNA conjugate as a template, transcription is performed using T7 RNA polymerase to form a corresponding RNA conjugate. Single-stranded DNA linkers (20, 40, 60, and 80 nucleotides in length) chemically synthesized are separately ligated to the single-stranded RNA conjugate using T4 RNA ligase. Next, a 25-residue single-stranded DNA-RNA chimeric oligonucleotide (21 residues for DNA and 4 residues for RNA) named peptide acceptor was added to this conjugate in the presence of single-stranded DNA. And ligate using T4 DNA ligase.

  The peptide acceptor sequence has the 3'-terminal sequence of alanyl-tRNA and promotes the incorporation of the puromycin derivative into the A site of the ribosome. Therefore, the peptide acceptor is located between the spacer region and the puromycin region. It is preferable to use an acceptor.

  By linking the previously chemically synthesized pdCpPur to the 3 'end of this conjugate with T4 RNA ligase, an RNA-DNA chimera conjugate that can be used as a cell-free protein synthesis gene, a 5'-T7 promoter region (RNA) -SD region (RNA) -4 repeat region (RNA) -spacer region (DNA) -peptide acceptor region-puromycin-3 'can be obtained.

  When protein synthesis is performed using the above-described cell-free protein synthesis system using the above RNA-DNA chimera conjugate as a gene, the nucleic acid part (RNA-DNA chimera conjugate; genotype) and the protein part (phenotype) become puromycin. A conjugate linked by a chemical bond can be obtained.

  In the above method, a chimeric spacer of DNA and polyethylene glycol can be used instead of a chimeric spacer of DNA and RNA.

  Furthermore, in the above method, a double-stranded spacer consisting of DNA-DNA double strand, RNA and short-chain (for example, about 15 to 25 nucleotides) PNA or DNA in place of the chimeric spacer of DNA and RNA. Can be used. The spacer consisting of DNA and DNA double-stranded does not need to be double-stranded over its entire length, and is mostly double-stranded (usually, several residues at both ends are single-stranded, and the other part is double-stranded. ). A double-stranded spacer consisting of RNA and short PNA or DNA is prepared by (a) preparing a DNA containing a gene without a stop codon and the base sequence of the spacer, and (b) transcribing the prepared DNA to RNA. (C) connecting a substance having a nucleoside or a nucleoside-like chemical structural skeleton, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, to the 3′-terminal side of the obtained RNA; (D) It can also be prepared by adding a short-chain PNA or DNA to the 3′-terminal side of the gene portion of the resulting RNA conjugate to form a double strand.

Unless otherwise specified, genetic engineering techniques such as nucleic acid isolation / preparation, nucleic acid ligation, nucleic acid synthesis, PCR, plasmid construction, and translation in a cell-free system are not described in Samrook et al.
al. (1989) Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press or a method analogous thereto.

  The assigning molecule of the present invention can also be obtained by sequentially linking each constituent element by a known chemical bonding method, in addition to the methods described above.

  As shown in FIG. 12, the protein evolution experiment method of the present invention comprises (1) construction of an in vitro virus genome, (2) completion of an in vitro virus, (3) selection process, (4) mutagenesis, 5) A method comprising the steps of: amplifying, and the functional protein can be modified and created by these steps or by repeating these steps as necessary. Among them, the steps (1) and (2) can be performed according to the construction method described in detail above. That is, the step (1) corresponds to the construction of a linked body of a nucleoside or a substance having a chemical structure skeleton similar to a nucleoside, and the step (2) corresponds to the construction of an associating molecule from the linked body. Equivalent to. Therefore, the steps (3), (4) and (5) will be described here.

  (3) The selection process refers to the step of evaluating the function (biological activity) of the protein part constituting the in vitro virus and selecting the in vitro virus based on the desired biological activity. Such steps are known and are described, for example, in Scott, JK & Smith, GP (1990) Science, 249, 386-390; Devlin, PE et al. (1990) Science, 249, 404-406; Mattheakis, LC et Natl. Acad. Sci. USA, 91, 9022-9026 and the like.

Next, in the steps of (4) mutagenesis and (5) amplification, a mutation is introduced into the nucleic acid portion of the selected in vitro virus, and amplification is performed by PCR or the like. Here, when the nucleic acid portion of the in vitro virus is RNA, the mutation may be introduced after synthesizing cDNA with reverse transcriptase, and the amplification of the nucleic acid portion may be performed while introducing the mutation. Mutagenesis was performed using the established Error-prone PCR (Leung, D.
W., et al., (1989) J. Methods Cell Mol. Biol., 1, 11-15) and Sexual PCR (Stemmer,
Natl. Acad. Sci. USA 91, 10747-10751).

  Using the nucleic acid portion of the in vitro virus with the mutation introduced and amplified, (1) constructing the in vitro virus genome, using it, (2) completing the in vitro virus, and (3) targeting the selection process (4) Mutagenesis and amplification can be performed by selecting according to the biological activity. By repeating these steps as necessary, it becomes possible to modify and create a functional protein.

  The respective means in the apparatus of the present invention for performing the above-described evolution experiment method are each known, and operations such as addition of reagents, stirring, temperature control, and evaluation of biological activity in these means are performed by methods known per se. Just do it. By combining these operations, a fully automatic or semi-automatic apparatus of the present invention can be constructed.

  In the protein-protein or protein-nucleic acid interaction assay method of the present invention, the construction step of constructing an assigning molecule generally comprises (1) synthesizing mRNA from a gene library or a cDNA library, And (2) using an cell-free protein synthesis system to construct an in vitro virus in which mRNA and its corresponding protein are linked on ribosomes.

  In the step (1), mRNA is synthesized using RNA polymerase from a cDNA containing a sequence having a known sequence and a cDNA containing a sequence fragmented with an appropriate restriction enzyme from a cDNA containing a sequence corresponding to the ORF, and This corresponds to constructing a viral genome in vitro.

  The steps of (1) construction of the in vitro virus genome and (2) construction of the in vitro virus can be performed according to the construction method described in detail above.

In addition, the assay step for examining the interaction between the assigning molecule and another protein or nucleic acid (DNA or RNA) generally involves a specific function among the in vitro viruses constructed in steps (3) and (2). And (4) reverse transcription, amplifying and selecting the sequence of the selected in vitro virus.

  In the step (3), the target protein, nucleic acid (DNA or RNA) and other substances such as carbohydrates and lipids are previously bound to microplates and beads via covalent or non-covalent bonds. Then, the in vitro virus constructed in step (2) is added, and the mixture is reacted at a certain temperature for a certain period of time, and then washed to remove in vitro virus that does not bind to the target. Thereafter, the in vitro virus bound to the target is released. This step can be carried out according to an established ELISA (Enzyme Linked Immunosorbent Assay) (Crowther, JR (1995) Methods in Molecular Biology, Vol. 42, Humana Press Inc.).

  In the step (4), the in vitro virus released in the step (3) is reversely transcribed and amplified by reverse transcription PCR, and the sequence of the amplified DNA is determined directly or after cloning.

According to the assay method of the present invention, (1) mRNA is synthesized from gene DNA having a known or unknown sequence,
constructing an in vitro virus genome, (2) using it to construct an in vitro virus, and (3) binding to target proteins or nucleic acids or other substances, such as carbohydrates and lipids, from in vitro viruses By selecting only (4) reverse transcription, amplification, cloning, and sequencing of the selected in vitro virus, it becomes possible to identify the function of a gene product (protein) corresponding to a gene whose function is unknown.

  In order to perform the above-described interaction assay method, an apparatus may be constructed by combining known appropriate means. The respective means in the present apparatus are known, and operations such as addition of reagents, stirring, temperature control, and evaluation of biological activity in these means may be performed by methods known per se. By combining these operations, an apparatus for performing a fully or semi-automated interaction assay method can be constructed.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as helping to obtain a specific understanding of the present invention, and the scope of the present invention is not limited by the following examples. It is not done.

Preparation of in vitro virus (1)
<1> Preparation of 3 'end of nucleic acid part (a) Synthesis of phosphorylated puromycin (pPur) Material: puromycin (3'-[α-Amino-p-methoxyhydrocinnamamido] -3'-deoxy-N, N '-dimethyl-adenosine) was purchased from Sigma. Phosphorous oxychloride and Trimethyl phosphate were purchased from Wako Pure Chemical.

Method: A solution obtained by mixing 1.5 mmol of phosphorus oxychloride and 11.4 mmol of trimethyl phosphate was cooled on ice, 0.3 mmol of puromycin was added, mixed well, and reacted at 0 ° C. for 7 hours (Yoshikawa, M. et al. (1969) Bull. Chem. Soc. Jap. 42, 3505-3508). Next, the reaction solution was added to a mixture of iced 40 ml of acetone, 20 ml of ether, and 0.4 g of sodium perchlorate (NaClO ₄ ), and the mixture was stirred well. Add 720 ml of water and stir all day and night at 4 ° C to hydrolyze chlorine groups. The hydrolyzed and precipitated product is separated by centrifugation and washed with acetone and ether. The white powder was dried under vacuum to obtain phosphorylated puromycin in 70-90% yield based on puromycin.

(B) Acetylation protection of phosphorylated puromycin Material: trifluoroacetic acid (TFA) was purchased from Nacalai Tesque. Trifluoroacetic anhydride (TFAA) was purchased from Wako Pure Chemical Industries.

  Method: 0.2 mmol of dried phosphorylated puromycin and 5 ml of TFA were mixed, and 2 ml of TFAA was added at −10 ° C. and stirred. The reaction was carried out for 1 hour while mixing at room temperature (Weygand, F. & Gieger, R. (1956) Chem. Ber. 89, 647-652). The reaction was stopped by adding 50 ml of water, and the procedure of adding water (10 ml) and evaporating to dryness under reduced pressure was repeated 5 times to remove TFA. Finally, add 50 ml of water and freeze-dry, and puromycin in which the amino group of the amino acid part of the puromycin and the 2 'hydroxyl group of the ribose part are protected with an acetyl group is 50-60% of the phosphorylated puromycin. In a yield of

(C) Synthesis of dCpPur (2'-Deoxycytidyl (3 '→ 5') puromycin) Material: BZ-DMT deoxycytidine (N4-Benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-deoxycytidine ) Was purchased from Sigma, and DCC (Dicyclohexyl carbodiimide) was purchased from Watanabe Chemical. Pyridine was purchased from Nacalai Tesque.

  Method: 40 μmol of phosphorylated puromycin protected with an acetyl group and 600 μmol of Bz-DMT deoxycytidine were dehydrated by adding pyridine (2 ml) and evaporating to dryness three times. Was added thereto with stirring, and 400 μmol of DCC was added thereto with stirring, and reacted at room temperature for 3 days to 2 weeks (Ralph, RK et al. (1965) J. Am. Chem. Soc. 87, 5661) -5670 and Harris, RJ et al. (1972) Can. J. Biochem. 50, 918-926). After the reaction, the mixture was reacted with 5 ml of 80% acetic acid for 2 hours to deprotect the DMT group. Next, the mixture was reacted with 6 ml of concentrated aqueous ammonia-ethanol (2: 1 by volume) at 20 ° C. for 2 days to deprotect the acetyl group. The concentrated aqueous ammonia was removed by evaporating under reduced pressure, and then dissolved in 40 ml of water. This solution is adsorbed through a column packed with QAE-Sephadex A-25 (Pharmacia), fractions containing the desired product are eluted with 0.5 M triethylamine carbonate (TEAB, pH 7.5), and then frozen. Dried, finally separated by HPLC and deprotected dCpPur was obtained in 1-5% yield based on puromycin.

<2> Preparation of nucleic acid part (in vitro virus genome)
Two types were prepared as in vitro virus genomes. That is, (1) for connecting a nucleic acid part and a protein part in a site-specific manner, and (2) for connecting a nucleic acid part and a protein part in a non-site-specific manner.

Materials: E. coli S30 Extract System for Linear Templates is purchased from Promega. T7 RNA polymerase, T4 DNA ligase, T4 DNA kinase, human placenta-derived ribonuclease inhibitor, EcoRI, BamHI, and deoxyribonucleotide were purchased from Takara Shuzo. Restriction enzymes BstNI and BglII were purchased from New England Labs. [ ³⁵ S] methionine and [γ- ³² P] ATP were from Amersham, and Taq DNA polymerase was from Kurabo Industries and Greiner. All other biochemical reagents were from Sigma and Wako Pure Chemical. The plasmid (pAR3040) incorporating the microtubule-binding region (4 repeats) of the human tau protein is obtained by using the PCR method to probe the full-length gene of the human tau protein from the human brain cDNA library cloned into λZAPII and incorporating the plasmid into the plasmid. Only the four repeat regions were amplified by PCR and incorporated into a plasmid. As a PCR (Polymerase chain reaction) apparatus, PTC-100 type (MJ Research) and ASTEC PC800 type (Astec) were used.

(1) Creation of a genome for site-specific binding A. Creation of DNA for 4 repeats
1) The microtubule region of human tau protein (Goedert, M. (1989) EMBO J. 8, 392-399)
A plasmid (pAR3040) incorporating the repeat) was constructed, and cut into a linear form by restriction enzymes BglII and BamHI.

  2) Four repeats containing the T7 promoter region and Shine-Dalgarno sequence were amplified from this genome by PCR. At this time, Left + (SEQ ID NO: 1) was used on the 5 ′ side and Right− (SEQ ID NO: 2) was used on the 3 ′ side. The Right- sequence also mutates leucine before the ocher stop codon to an amber stop codon. PCR conditions were repeated 30 times with denaturation at 92 ° C / 30 seconds, annealing at 65 ° C / 30 seconds, and extension reaction at 73 ° C / minute.

  3) Next, after purifying this amplified genome, mutation was added using PCR to increase the incorporation of methionine and enhance the detection of radioisotopes. That is, primers Left- (SEQ ID NO: 3) and Right + (SEQ ID NO: 4) containing the region to be mutated are synthesized, and the DNA of 2) above is used as a template, and first, primers Left + and Left- are amplified by PCR, The amplified DNA was designated as "Left". In addition, amplification was performed by PCR with primers Right + and Right-, and the amplified DNA was designated as “Right”. "Left" and "Right" were cut out from the gel and extracted by 5% acrylamide denaturing gel electrophoresis. The excised Left and Right were first amplified by PCR under the conditions described above without primers. Further, 1 μl was collected from this reaction solution as a template, and amplified by PCR under the same conditions with the primers Left + and Right−. As a result, DNA of the mutant 4 repeat portion in which the number of methionine was increased from 1 to 4 was created.

B. Ligation of alanine suppressor tRNA (Ala · sup tRNA) with spacers of various lengths to four repeats
1) The BamH1 site at the 3 'end of the 4-repeat portion of A above was digested with BamH1. Then, to remove the 3 'fragment of the BamH1 site, only the 4 repeats on the 5' side were extracted and purified using a QIAquick PCR Purification Kit (manufactured by QIAGEN).

  2) Spacer-A (SEQ ID NO: 5) phosphorylated with T4 kinase on the 5 ′ side of the purified product of 1) above was backed by Spacer-B (SEQ ID NO: 6), and ligated with T4 DNA ligase.

  3) Spacer-C (SEQ ID NO: 7) phosphorylated by T4 kinase and Spacer-B having a region complementary thereto were ligated using T4 DNA ligase. The reaction was performed at 15 ° C. for 2 hours. Thereafter, purification was performed by ethanol precipitation.

  4) Dissolve the products of 2) and 3) above and Spacer-D (SEQ ID NO: 8) and the 5′-phosphorylated sup tRNA (SEQ ID NO: 9) in T4 DNA ligase buffer (Buffer), and heat them at 85 ° C. for 2 minutes. After cooling with ice, cool on ice. Further, T4 DNA ligase was added and reacted at 15 ° C. for 2 hours. After phenol extraction, ethanol precipitation was performed.

  5) Using the product obtained in 4) above as a template, using primer Left + and primer 3'Pur- (SEQ ID NO: 10), denaturation 92 ° C / 30 seconds, annealing 65 ° C / 30 seconds, extension reaction 73 ° C / 1 minute. The product was amplified by PCR under conditions of 30 times, and the product was subjected to acrylamide denaturing gel electrophoresis to cut out three regions A, B, and C having different migration distances, and DNA was extracted.

  6) Using A, B, and C having different lengths of 5) as templates as templates, the DNA was again amplified by PCR under the same conditions, and its length was identified by electrophoresis, and at the same time, it was used as a template DNA for transcription. Thus, it was found that the fraction c had 0 to 5 Spacer-C, the b fraction had 6 to 14, and the a fraction had 15 to 18 inserted.

C. Preparation of RNA genome and ligation of dCpPur The A, B, and C regions obtained in B above were transcribed into RNA by reacting at 37 ° C. for 2 hours using T7 polymerase. Further, the dCpPur obtained in the preparation of the 3 ′ end of the <1> nucleic acid portion was reacted with T4 polynucleotide kinase in the presence of ATP at 15 ° C. for 24 hours to phosphorylate, and then the transcribed RNA genome and T4 RNA ligase were reacted. The reaction was performed at 4 ° C. for 50 hours. By this operation, an RNA genome having sup tRNA with puromycin at the 3 ′ end was constructed.

(2) Preparation of genome for site-specific binding A. Preparation of DNA and RNA of mutant 4 repeat portion DNA of mutant 4 repeat portion is basically prepared by the same method as A in (1) above. did. However, a new primer New / Right- (SEQ ID NO: 10) was used in order to eliminate the termination codon by replacing the two stop codons, ie, amber with glutamine and ocher with lysine, and to make the 3 ′ end purine-rich. It was synthesized and amplified by PCR under the conditions of denaturation at 92 ° C / 30 seconds, annealing at 65 ° C / 30 seconds, and extension reaction at 73 ° C / minute for 30 times with Left +. Using this DNA as a template, a reaction was performed at 37 ° C. for 2 hours using T7 polymerase to obtain an RNA genome.

B. Ligation of Spacer 1 to 4 DNA consisting of 21 bases, Spacer 1 (SEQ ID NO: 11), DNA consisting of 40 bases, Spacer 2 (SEQ ID NO: 12), DNA consisting of 60 bases, Spacer 3 (SEQ ID NO: 13) ), Spacer4 (SEQ ID NO: 14) consisting of 80 bases was reacted with T4 polynucleotide kinase at 36 ° C for 1 hour, and then reacted with T4 RNA ligase at 10 ° C for 48 hours.

C. Linkage of peptide acceptor (P-Acceptor)
Synthesizes a chimeric nucleic acid consisting of 21 bases of DNA and 4 bases of RNA, a total of 25 bases, and a peptide acceptor (P-Acceptor) (SEQ ID NO: 15) for the purpose of binding dCpPur to the 3 'end and increasing the efficiency of incorporation into ribosomes. did. After phosphorylating the 5 'end of P-Acceptor with T4 polynucleotide kinase at 36 ° C for 1 hour, it was backed by Back3' (SEQ ID NO: 16) having a complementary sequence, and prepared in B above. The 3 'end of each of the spacers was ligated by performing a reaction at 16 ° C. for 2 hours using T4 DNA ligase. In addition, this P-Acceptor was directly ligated to the 3 ′ end of the RNA obtained in the above A by reacting it at 10 ° C. for 48 hours using T4 RNA ligase, and this was linked to the Non-Spacer genome. Name.

D. Ligation of dCpPur dCpPur obtained in the above <1> Preparation of 3 ′ end of nucleic acid portion was reacted at the 3 ′ end of each genome prepared in C above at 15 ° C. for 24 hours using T4 polynucleotide kinase, and phosphorylated. After oxidation, the reaction was carried out at 4 ° C. for 50 hours using T4 RNA ligase. As a result, a chimeric RNA genome with puromycin at the 3 ′ end was constructed.

<3> Optimization of nucleic acid part Site-specific method The E. coli cell-free translation system of E. coli together with biotinylated lysine-tRNA (Promega) was used for each of the RNA genomes classified into the lengths of the a, b, and c fractions prepared in (1) of <2> above. After translation with [E. coli S30 Extract Systems for Linear Templates (Promega)], add 5 mg of magnetic particle dynabeads (Dinal) with streptavidin to each tube, and incubate at room temperature for 1 hour. Next, the dynabeads are collected by a magnet and the supernatant is aspirated. The remaining dynabeads were washed twice with 1000 μl of B & W Buffer. Further, after washing twice with 500 μl of RT-PCR Buffer, the cells are suspended again with 500 μl of RT-PCR Buffer. 50 μl thereof was collected and transferred to a 500 μl Eppendorf tube, dynabeads were fixed with a magnet, and the supernatant was sucked. Add RT-PCR Buffer, reverse transcriptase and Taq polymerase [Access RT-PCR System (Promega)] to the remaining Dynabeads, reverse transcription at 48 ° C for 1 hour, PCR at 94 ° C / 30 seconds, PCR at 65 ° C / 40 seconds The primers were Right + (SEQ ID NO: 4) and 3'Pur- (SEQ ID NO: 10) at 68 ° C. for 1 minute and 40 seconds, 40 times. FIG. 5 shows the fractions a, b, and c examined by electrophoresis.

  Here, a band was detected from the group of the fraction c (lane 3 in FIG. 5). This band was separated from the gel by electrophoresis and further ligated by PCR to "Left" having a T7 promoter and Shine-Dalgarno region, which was further ligated with primers Left + (SEQ ID NO: 3) and 3'Pur- (SEQ ID NO: 10 ) Was amplified by PCR. This genome was named "Stranger".

Next, to determine whether the translated protein actually binds to the mRNA (RNA genomic part), this Stranger ligates pdCpPur to the 3 'side with T4 RNA ligase after transcription, and The 'side was dephosphorylated with HK phosphatase (Epicentre) at 30 ° C for 1 hour, and labeled with T4 polynucleotide kinase in the presence of [γ- ³² P] ATP. This was added to the E. coli cell-free translation system as mRNA, and reacted at 37 ° C. for 1 hour and 40 minutes. FIG. 6 shows the result of electrophoresis of this by 18% SDS-PAGE. This indicates that the nucleic acid part (genotype) and the protein part (phenotype) bind at a ratio of about 80% or more, and an in vitro virus, that is, a genotype-phenotype associating molecule is formed.

B. Site non-designated manner already for those short spacer site-directed methods have been chosen, the 3 'end of the "Non-location spacer" RNA genome without spacers, using T4 polynucleotide kinase [γ- ³² P] In the presence of ATP, 5C phosphorylated dCpPur was reacted with T4 RNA ligase at 4 ° C. for 50 hours to ligate. Next, this was added to an Escherichia coli cell-free translation system together with the usual mRNA encoding 4 repeats, and reacted at 37 ° C. for 1 hour and 30 minutes. 10 μl of this reaction was digested with ribonuclease T2, and an equivalent amount of the reaction solution was electrophoresed on 18% SDS-PAGE and analyzed with an image analyzer BAS2000 (Fuji Film) (FIG. 7).

As a result, the RNA was degraded by ribonuclease T2 only in the protein portion, and thus a band appeared at the same moving distance as the four repeat protein portion labeled with [ ³⁵ S] methionine as a control. On the other hand, when no treatment was performed, it was found that the molecular weight was clearly higher than the protein portion of the four repeats. Also, this is not the mRNA itself (about 400 bases) that is more mobile than the tRNA. Therefore, it was identified that RNA and protein were bound. That is, this result indicates that the nucleic acid portion and the protein portion were linked in a site-nonspecific manner.

Preparation of in vitro virus (2)
<1> Preparation of 3 ′ end of nucleic acid part (a) Synthesis of rCpPur (ribocytidyl (3 ′ → 5 ′) puromycin) Material: puromycin is obtained from Sigma, and rC-beta amidite (N4-benzoyl-5 ′) -O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyl) -cytidine-3'-O- [O- (2-cyanoethyl) -N, N'-diisopropyl-phosphoramidite) Tetrazole was purchased from Nippon Millipore, Tetrabutylammonium Fluoride from Aldrich, QAE-Sephadex from Pharmacia, and silica gel for chromatography from Merck from Perceptive.

Method: Puromycin (50 mg, 92 μmol) was dissolved in 2 ml of dry pyridine, evaporated under reduced pressure and dried. This operation was repeated three times. To this was added 15 ml of a 4% tetrazole / acetonitrile solution and allowed to stir at room temperature. The reaction was monitored by silica gel thin-layer chromatography (TLC, developing solvent: chloroform: methanol = 9: 1). Usually, the reaction is completed in one day. After the reaction, the solvent was expelled under reduced pressure, 3 ml of a solution of 0.1 M iodine dissolved in tetrahydrofuran / pyridine / water = 80: 40: 2 was added, and the resulting phosphite-triester was oxidized while stirring at room temperature. I let it. After one and a half hours, the solvent was driven off under reduced pressure, and the remainder was extracted with chloroform. After the extract was dried in the presence of anhydrous magnesium sulfate, the solvent was driven off under reduced pressure. This was subjected to silica gel column chromatography, and eluted with chloroform / methanol = 90: 10. Ribocytidyl puromycin (CpPur) with a protecting group is eluted at Rf 0.32 by TLC on silica gel (developing solvent: chloroform: methanol = 9: 1). Next, the protecting group was deprotected. The protected ribocytidyl puromycin was first treated with 0.5 ml of an 80% aqueous acetic acid solution at room temperature for 1 hour, acetic acid was removed under reduced pressure, and then 0.5 ml of a mixed solution of concentrated aqueous ammonia / ethanol = 2: 1 was added. added. After leaving at room temperature for 15 hours, the solvent was driven off under reduced pressure, and 0.5 ml of a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran was added to remove the β-cyanoethyl group. After 30 minutes, the solvent was driven off under reduced pressure, and the residue was subjected to QAE-Sephadex column chromatography, eluting with a linear gradient of 0-0.5 M triethylamine carbonate. The eluate was collected and lyophilized. 10 mg of ribocytidyl puromycin was obtained. The fact that the synthetic product is ribocytidyl puromycin means that cytidine and puromycin-5'-phosphate can be obtained in equal amounts by nuclease P1 digestion, and that [M + H] ⁺ molecules are obtained by MALDI / TOF mass spectrometry. The ion was identified by its appearance at m / z 777.

<2> Nucleic acid part (preparation of in vitro virus genome)
Material: Rabbit reticulocyte lysate cell-free protein synthesis system was purchased from Promega. T7 RNA polymerase, T4 DNA ligase, T4 RNA ligase, T4 polynucleotide kinase, human placenta-derived ribonuclease inhibitor, EcoRI, BamHI, and deoxyribonucleotide were purchased from Takara Shuzo. Restriction enzymes BstNI and BglII were purchased from New England Labs. [ ³⁵ S] methionine and [ ³² P] -γATP were from Amersham, and Taq DNA polymerase was from Kurabo Industries and Greiner. All other biochemical reagents were from Sigma and Wako Pure Chemical. A plasmid (pAR3040) incorporating the N-terminal half region of human tau protein (amino acid residues 1-165) was used to probe the full-length gene for human tau protein from the human brain cDNA library cloned into λZAPII by PCR. Then, only the N-terminal half region from the plasmid integrated was amplified by PCR and integrated into the plasmid. As a PCR (Polymerase chain reaction) apparatus, ASTECPC800 type (Astec) was used.

(1) Preparation of genome Preparation of N-terminal half-region DNA
An mRNA encoding the N-terminal half-region of human tau protein (amino acid residues 1-165) with or without a spacer, peptide acceptor, rCpPur-linked stop codon at the 3 'end was constructed as follows (No. 8).

  1) A plasmid (pAR3040) incorporating the N-terminal half region of human tau protein (Goedert, M. (1989) EMBO J. 8, 392-399) is cut into a linear form with the restriction enzyme BglII.

  2) Amplify the N-terminal half region (amino acid residues 1-165) from this genome by PCR. At this time, Left1 (SEQ ID NO: 18) was used on the 5 'side and Right1 (SEQ ID NO: 19) containing a stop codon and Right2 (SEQ ID NO: 20) containing no stop codon were used on the 5' side. PCR conditions were repeated 30 times with denaturation at 92 ° C / 30 seconds, annealing at 65 ° C / 30 seconds, and extension reaction at 73 ° C / minute.

  3) DNA (SEQ ID NO: 21) in which a promoter region of T7 RNA polymerase, a Kozak sequence, and a DNA sequence corresponding to amino acid residues 1 to 25 of human tau protein were connected in this order was prepared by chemical synthesis.

4) The two purified DNAs obtained by the above operations 2) and 3) were ligated by the following two-step PCR. That is, the mixture of the two DNAs was first amplified in the absence of a primer, and then amplified in the presence of the Left2 (SEQ ID NO: 22) and Right1 (SEQ ID NO: 19) or Right2 (SEQ ID NO: 20) primers. By the above operation, a DNA having a T7 RNA polymerase promoter and a Kozak sequence upstream of the ORF in the N-terminal half region of the human tau protein was prepared. Using this DNA as a template, reaction was carried out at 37 ° C. for 2 hours using T7 RNA polymerase to obtain RNA.

B. Linking Spacer and peptide acceptor
A chimeric nucleic acid consisting of Spacer5 (SEQ ID NO: 23), 21 bases of DNA and 4 bases of RNA, a total of 25 bases, and a peptide acceptor (P-Acceptor) (SEQ ID NO: 15) were chemically synthesized. After a reaction at 36 ° C. for 1 hour using T4 polynucleotide kinase to phosphorylate the 5 ′ end of the peptide acceptor, it was backed by splint DNA (SEQ ID NO: 24) having a sequence complementary thereto, and The reaction was ligated at 16 ° C. for 2 hours using T4 DNA ligase at the 3 ′ end.

C. Ligation of RNA and Spacer-peptide acceptor To phosphorylate the 5 'end of the Spacer5-peptide acceptor conjugate obtained in B above, use T4 polynucleotide kinase and react at 36 ° C for 1 hour. Ligation was performed by reacting the RNA obtained in A above with T4 RNA ligase at 4 ° C. for 48 hours.

D. lCpPur ligation rCpPur obtained by preparation of <1> 3 ′ end of nucleic acid portion at the 3 ′ end of the genome created in C above was reacted with T4 polynucleotide kinase at 15 ° C. for 24 hours to phosphorylate, The reaction was performed at 37 ° C. for 30 minutes using RNA ligase. As a result, a chimeric RNA genome having puromycin at the 3 ′ end was constructed.

E. FIG. RCpPur binding to the N-terminal half-terminal C-terminus of human tau protein To effectively link the C-terminus of the protein to the RNA encoding it, the distance between puromycin and the stop codon and the presence or absence of a stop codon are important It is considered to be a factor. Therefore, the following three genomes were prepared in order to examine the effects of these factors on the intermolecular binding. That is, the mRNA encoding the N-terminal half (1-165) of the human tau protein has (1) a stop codon but no DNA spacer at the 3 'end, and (2) both a stop codon and a DNA spacer. And (3) those without a stop codon but with a DNA spacer. From these three genomes, protein synthesis was carried out in the presence of ³² P-labeled rCpPur in a cell-free translation system using rabbit reticulocyte extracts (FIG. 9). When no DNA spacer was attached to the 3 'end, it was found that rCpPur ligated to the C-terminus of the protein with the same efficiency regardless of the presence or absence of a stop codon. That is, the bands of the protein to which rCpPur was linked (the first and second lanes from the left in FIG. 9) were the monomers of the protein (the first lane) labeled with [ ³⁵ S] methionine by SDS-PAGE (SDS-polyacrylamide electrophoresis). (The rightmost lane in FIG. 9). On the other hand, it was found that rCpPur ligated to the C-terminus of the protein with about three times the efficiency of the former two if the DNA spacer was attached without the stop codon (third lane from the left in FIG. 9). . This result indicates that the translational pause of the ribosome occurs on the DNA sequence, and as a result, rCpPur and the protein are efficiently linked. In addition, this result indicates that when a genome without a stop codon and with a DNA spacer and rCpPur at the 3 'end was used as mRNA in a cell-free translation system, puromycin at the 3' end of the mRNA would produce the corresponding translated protein. This suggests that it can be efficiently bound to the C-terminus.

<3> Construction of in vitro virus in cell-free translation system mRNA encoding N-terminal half (1-165) of human tau protein constructed in section <2> Nucleic acid part (preparation of genome of in vitro virus) The genome consisting of -DNA spacer (105 mer) -peptide acceptor-rCpPur was translated using rabbit reticulocyte extract. First, using RNA encoding the N-terminal half (1-165) of human tau protein as mRNA, and examining the incorporation of [ ³⁵ S] methionine into the protein, the N-terminal half (1-165) monomer (~ Bands appeared at 28 kDa) and the dimer (~ 55 kDa). In this case, the monomer is predominant, and the dimer is negligible (the leftmost lane in FIG. 10A). This result indicates that RNA encoding the N-terminal half (1-165) of human tau protein functions as mRNA. A genome consisting of mRNA-DNA spacer (105mer) -peptide acceptor-rCpPur encoding the N-terminal half (1-165) of human tau protein is translated by a cell-free translation system containing the same [ ³⁵ S] methionine, Examining the time sequence (5, 10, 20, and 40 minutes), we found that in addition to the monomer and dimer positions, a new broad band was located in the genome (the rightmost lane in Fig. 10 (A)). Appeared a little above the position. The intensity of this band increased with the passage of reaction time (second to fifth lanes from the left in FIG. 10 (A)) and increase in the amount of genome (lanes 3 and 4 in FIG. 10 (B)). . This result indicates that the genome was covalently linked to the C-terminus of the protein via puromycin. This also means that the genotype was covalently linked to the phenotype. In other words, a genotype-phenotype mapping molecule was created. The present inventors have named this assigning molecule an in vitro virus. When the effect of the length of the DNA spacer on the formation of the virus in vitro was examined, it was found that the formation was not efficient with a length of about 80 mer, and a length of at least 100 mer was required.

Further confirmation of in vitro virus production was made using rCpPur labeled with ³² P. That is, a genome comprising mRNA-DNA spacer (105 mer) -peptide acceptor- [ ³² P] rCpPur encoding the N-terminal half (1-165) of human tau protein was translated using a rabbit reticulocyte extract. Genome-protein binding was confirmed by digestion with mung bean nuclease. That is, when the translation product (lane 3 in FIG. 11) was digested with peanut nuclease, the translation product was located at a position corresponding to the monomer and dimer of the N-terminal half (1-165) of human tau protein (lane 1 in FIG. 11). A band appeared (lane 4 in FIG. 11). This means that the C-terminus of the protein has rCpPur labeled with ³² P. This result also indicates that the genome was covalently linked to the C-terminal of the protein via puromycin. The efficiency of conjugation was estimated to be about 10%. Since in vitro virus genomes at concentrations of 40 to 100 pmol / ml can be prepared, the in vitro virus produced consists of a population containing 2.4 to 6 × 10 ¹² mutants, the number of which is determined by the phage display method (Scott, JK). & Smith, GP (1990) Science 249, 386-390). Mapping genotypes and phenotypes using in vitro viruses has advantages such as avoidance of permeability problems and the introduction of various unnatural amino acids. Enables production of various functional proteins.

Experimental method for protein evolution using in vitro virus
As shown in FIG. 12, protein evolution experiment methods using in vitro virus include (1) construction of in vitro virus genome, (2) completion of in vitro virus, (3) selection process, (4) It consists of the steps of mutagenesis and (5) amplification, and enables modification and creation of functional proteins. In particular, by repeatedly performing this step, it is possible to efficiently modify and create a functional protein. Among them, the steps (1) and (2) have been specifically described in Examples 1 and 2 above. Here, the steps (3), (4) and (5) will be described.

First, it was examined whether or not peptides specific to the antibody were eliminated. Specifically, mouse IgG is used as an antibody, and the peptide sequence that specifically binds to the antibody is a known ZZ region of protein A (Nilsson, B., et al., (1987) Protein Eng., 1, 107-113). ) Was used. The N-terminal region (1-105) of human tau protein (Goedert, M. (1989) EMBO J. 8, 392-399) was used as a control. According to the in vitro virus construction method described in Examples 1 and 2, an in vitro virus genome encoding the ZZ region of protein A and the N-terminal region (1-105) of human tau protein was prepared. The ratio of the in vitro virus genome containing the ZZ region of protein A to the in vitro virus genome containing the N-terminal region (1-105) of human tau protein is 1: 1, 1:10, 1: 100, 1: 1000, etc. And translated in a cell-free translation system using a rabbit reticulocyte extract at 30 ° C. for 10 minutes. Thereafter, the translation product was diluted, centrifuged to remove the insoluble fraction, and the supernatant was added to a mouse IgG-adsorbed microplate (blocked with bovine serum albumin) and allowed to stand at 4 ° C. for 2 hours. Was placed. Thereafter, the translation product was removed from the microplate, and the plate was washed with a washing buffer (50 mM Tris acetic acid, pH 7.5 / 150 mM salt / 10 mM EDTA / 0.1% Tween 20) six times in total. , PH 2.8). The eluate was precipitated with ethanol and dissolved in 20 μl of sterile water to use as a template for reverse transcription PCR. Reverse transcription PCR uses reverse transcriptase (Avian Mieloblastosis Virus Reverse Transcriptase, manufactured by Promega), DNA polymerase (Tfl DNA Polymerase, manufactured by Promega), and RT + (SEQ ID NO: 25) and RT- (SEQ ID NO: 26) as primers. After reacting at 40 ° C. for 40 minutes, reverse transcriptase was inactivated by treatment at 94 ° C. for 5 minutes, and then a cycle of 94 ° C. for 30 seconds, 66 ° C. for 40 seconds, and 72 ° C. for 40 seconds was repeated 30 times. . The obtained PCR product was subjected to electrophoresis at 55 ° C. using a 4% polyacrylamide gel containing 8 M urea, and confirmed by silver staining. As a result, it was found that the in vitro virus genome containing the ZZ region of protein A can be amplified even by a hundredth of the in vitro virus genome containing the N-terminal region (1-105) of human tau protein as a control genome. This result indicates that the in vitro virus genome containing the ZA region of protein A specifically bound to mouse IgG via the translated ZZ region of protein A. Therefore, it was revealed that in vitro virus can be selected. Mutagenesis and amplification are already established in Error-prone PCR (Leung, DW, et al., (1989) J. Methods Cell Mol. Biol., 1,
11-15) and Sexual PCR (Stemmer, WPC (1994) Proc. Natl. Acad. Sci. USA 91, 10747-10751). Therefore, it was proved that the protein evolution experiment method using the in vitro virus shown in FIG. 12 was feasible.

It is a figure which shows the correspondence strategy of a genotype (nucleic acid part) and a phenotype (protein part). BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the construction method of the genotype and phenotype associating molecule of this invention of which nucleic acid part and protein part are site-specific. FIG. 3 is a diagram showing a chemically modified portion at the 3 ′ end of a nucleic acid portion, which is a point of construction of a genotype-phenotype associating molecule (in vitro virus). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a method for constructing a genotype-phenotype associating molecule of the present invention, in which a nucleic acid part and a protein part are site-specific. It is an electrophoresis photograph which shows optimization of a spacer in a site-specific method. After translating the prepared RNA genomes with spacers with the lengths of the a, b, and c fractions together with biotinylated lysine-tRNA in an Escherichia coli cell-free translation system, they are specifically adsorbed to magnetic particles with streptavidin. This is the result of 4% polyacrylamide electrophoresis (in the presence of 8 M urea) of reverse transcribed and DNA amplified by PCR (staining is silver stained). Lane 1 is the spacer length of the fraction a (residues 255-306), lane 2 is the spacer length of the b fraction (residues 102-238), and lane 3 is the spacer length of the c fraction. (0-85 residues). It is an electrophoresis photograph which shows connection of a nucleic acid part and a protein part by a site-specific method. Lane 1 is a translation product obtained by labeling mRNA encoding the 4 repeat region of tau protein with [ ³⁵ S] methionine in an Escherichia coli cell-free translation system, and lane 2 is a sup tRNA having puromycin at the 3 ′ end of the mRNA. This is the result of 18% polyacrylamide electrophoresis (8 M urea, in the presence of SDS) of a product obtained by ligation, labeling the 5 ′ end of this mRNA with [ ³² P], and translating with an E. coli cell-free translation system. It is an electrophoresis photograph which shows connection of a nucleic acid part and a protein part by a site non-specific method. Lane 1 is a translation product obtained by labeling mRNA encoding the four repeat region of tau protein with [ ³⁵ S] methionine in an E. coli cell-free translation system, and lane 2 is a 5′-terminal fragment of the mRNA via a DNA spacer at the 3 ′ end. Lane 3 is a product obtained by translating an mRNA linked to puromycin labeled with [ ³² P] with a cell-free translation system of Escherichia coli. Lane 3 is a product obtained by digesting the translation product of lane 2 with ribonuclease T2. Presence). FIG. 2 is a diagram showing an example of a method for constructing a genotype-phenotype associating molecule (in vitro virus) of the present invention. It is an electrophoresis photograph which shows binding of rCpPur to the C-terminal of the N-terminal half (1-165) of human tau protein. At the 3 'end of the mRNA encoding the N-terminal half (1-165) of the three genomes, human tau protein, one having a stop codon but no DNA spacer (the leftmost lane), the stop codon and the DNA spacer those without them (second lane from the left), a stop codon but has no built those having DNA spacers (third lane from the left), respectively, rabbit rCpPur presence labeled with ³² P Translation was performed at 30 ° C. for 20 minutes in a cell-free translation system using a reticulocyte extract. Translation products were analyzed on a 11.25% SDS-PAGE. The rightmost lane is a translation of mRNA encoding the N-terminal half (1-165) of human tau protein in the presence of [ ³⁵ S] methionine under the same conditions as above. 4 is an electrophoretic photograph showing the generation of an in vitro virus in a cell-free translation system. (A) shows the time course of in vitro virus production. N-terminal half of human tau protein (1-165) mRNA-DNA spacer (105 mer) encoding - free cells using rabbit reticulocyte extract containing a genome consisting of a peptide acceptor -rCpPur the ^[35 S] methionine The translation was performed by a translation system, and the translation product was examined at 30 ° C. over time (5 minutes, 10 minutes, 20 minutes, and 40 minutes). Translation products were analyzed on a 11.25% SDS-PAGE. The leftmost lane shows the incorporation of [ ³⁵ S] methionine into the protein under the same conditions as above, using RNA encoding the N-terminal half (1-165) of human tau protein as mRNA. The rightmost lane is the in vitro virus genome labeled with ³² P. (B) shows the effect of in vitro virus genome concentration on in vitro virus production. Lane 1 is the 3 ′ end of the genome labeled with [ ³² P] rCpPur, lane 2 is the genome (1.2 μg) with rCpPur attached to the 3 ′ end, and lane 3 is the 3 ′ end of the genome (0.33 μg) The lane 4 has rCpPur attached to the 3 ′ end of the genome (0.64 μg). In lanes 2 to 4, the genome was translated at 30 ° C. for 20 minutes using a cell-free translation system using rabbit reticulocyte extract containing [ ³⁵ S] methionine. Translation products were analyzed on a 11.25% SDS-PAGE. 4 is an electrophoretic photograph showing the generation of an in vitro virus in a cell-free translation system. An in vitro virus genome consisting of mRNA-DNA spacer (105 mer) -peptide acceptor- [ ³² P] rCpPur encoding the N-terminal half (1-165) of human tau protein was purified using rabbit reticulocyte extract by 30 Translated at 20 ° C. for 20 minutes. Translation products were analyzed on a 11.25% SDS-PAGE. Genome-protein binding was confirmed by digestion with mung bean nuclease. When the translation product (lane 3) was digested with peanut nuclease, a band appeared at a position corresponding to the monomer and dimer (lane 1) of the N-terminal half (1-165) of human tau protein (lane 4). Lane 2 is the in vitro viral genome labeled with ³² P. It is a figure which shows the process of the protein evolution experiment method using an in vitro virus.

Claims (56)

(A) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having the following structure: (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, and A desired protein, comprising: a step of constructing an assigning molecule, which comprises linking a nucleic acid part and a translation product of the gene; and a step of selecting out the assigning molecule obtained in the constructing step. How to choose. (A) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having the following structure: (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, and A step of constructing an assigning molecule characterized by linking a nucleic acid part and a translation product of the gene, a selecting step of selecting the assigning molecule obtained in the constructing step, and an assigning molecule selected by the selecting step An amplification step of amplifying the gene portion of the above, and subjecting the DNA obtained in the amplification step to a construction step, whereby the construction step, the selection step, and the amplification step are repeatedly performed. How to select proteins. (A) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having the following structure: (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, and A step of constructing an assigning molecule characterized by linking a nucleic acid part and a translation product of the gene, a selecting step of selecting the assigning molecule obtained in the constructing step, and an assigning molecule selected by the selecting step A method for obtaining DNA encoding a desired protein, comprising: 4. The method for obtaining DNA according to claim 3, wherein the DNA obtained in the amplification step is subjected to a construction step, whereby the construction step, the selection step, and the amplification step are repeated. (A) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having the following structure: (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, and A desired protein, comprising: a step of constructing an assigning molecule, which comprises linking a nucleic acid part and a translation product of the gene; and a step of selecting out the assigning molecule obtained in the constructing step. Method for selecting RNA that encodes (A) DNA containing a gene is prepared, (b) the prepared DNA is transcribed into RNA, and (c) an amino acid or a chemical structural skeleton similar to an amino acid via a spacer at the 3 ′ end of the obtained RNA. A substance having a chemical structural skeleton similar to a nucleoside or a nucleoside which can be covalently bonded to a substance having the following structure: (d) performing protein synthesis in a cell-free protein synthesis system using the obtained conjugate as mRNA, and A step of constructing an assigning molecule characterized by linking a nucleic acid part and a translation product of the gene, a selecting step of selecting the assigning molecule obtained in the constructing step, and an assigning molecule selected by the selecting step An amplification step of amplifying the gene portion of the above, and subjecting the DNA obtained in the amplification step to a construction step, whereby the construction step, the selection step, and the amplification step are repeatedly performed. A method for selecting RNA encoding a protein. The method according to any one of claims 1 to 6, wherein the DNA containing the gene further contains an expression control region. The method according to claim 1, wherein the spacer comprises a polymer. The method according to any of the preceding claims, wherein the length of the spacer is in the range of 100-1000 °. The method according to any one of claims 1 to 9, wherein the spacer comprises a nucleic acid. The nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or The method according to claim 10, which is a double strand of a single strand consisting of DNA and a short strand of DNA. The method according to claim 1, wherein the spacer comprises polyethylene glycol. The method according to claim 12, wherein the molecular weight of the polyethylene glycol is 3,000 to 30,000. 14. The method according to claim 9, wherein the spacer comprises polyethylene glycol and DNA. The method according to any one of claims 1 to 14, wherein the nucleoside or the substance having a chemical structure skeleton similar to the nucleoside is puromycin or a derivative thereof. The method according to any one of claims 1 to 15, wherein the linkage between the nucleic acid portion containing the gene and the translation product of the gene is formed by a covalent bond. 17. The method according to claim 16, wherein the covalent bond is an amide bond. The method according to any one of claims 1 to 17, wherein the assigning molecule selected in the selection step is selected using an interaction with a substance as an index. 19. The method according to claim 18, wherein the selection step includes a step of separating a complex due to an interaction between the substance bound to the solid phase and the assigning molecule obtained in the construction step. The method according to any one of claims 1 to 19, wherein the gene is an antibody or a partial gene thereof. The method according to any one of claims 1 to 19, wherein the gene is an enzyme or a partial gene thereof. The method according to any one of claims 1 to 21, wherein the DNA containing the gene is a library comprising a plurality of DNAs having different genes. The method according to any one of claims 1 to 22, wherein the DNA containing the gene comprises a random base sequence. A molecule in which a spacer made of a high molecular substance is bonded to the 3 'end of RNA having a gene. 25. A molecule having a nucleoside or a substance having a chemical structural skeleton similar to a nucleoside, which can be covalently bonded to an amino acid or a substance having a chemical structural skeleton similar to an amino acid, to the 3'-terminal side of the molecule according to claim 24. The molecule according to claim 24 or 25, wherein the RNA further comprises an expression control region. 27. The molecule according to any of claims 24 to 26, wherein the length of the spacer is in the range of 100-1000 °. 28. The molecule according to any of claims 24 to 27, wherein the spacer comprises a nucleic acid. The nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or 29. The molecule according to claim 28, which is a double strand of a single strand consisting of DNA and a short strand of DNA. 30. The molecule according to any one of claims 24-29, wherein the spacer comprises polyethylene glycol. 31. The molecule according to claim 30, wherein the spacer comprises polyethylene glycol and DNA. 32. The method according to claim 30, wherein the polyethylene glycol has a molecular weight of 3,000 to 30,000. 33. The molecule according to any one of claims 25 to 32, wherein the nucleoside or the substance having a chemical structure skeleton similar to the nucleoside is puromycin or a derivative thereof. The molecule according to any one of claims 24 to 33, wherein the gene is an antibody or a partial gene thereof. The molecule according to any one of claims 24 to 33, wherein the gene is an enzyme or a partial gene thereof. The molecule according to any one of claims 24 to 33, wherein the gene comprises a random base sequence. The library according to any one of claims 24 to 35, comprising a plurality of molecules having different genes. A molecule in which a nucleic acid portion having a nucleotide sequence reflecting a genotype is directly covalently bonded to a protein portion containing a protein encoded by the nucleotide sequence reflecting the genotype, wherein the nucleic acid portion includes DNA. 39. The molecule according to claim 38, wherein the nucleic acid portion further comprises an expression control region. 40. The molecule according to claim 38 or 39, wherein the DNA contained in the nucleic acid portion is a double strand of DNA and RNA. The molecule according to claim 38 or 39, wherein the DNA contained in the nucleic acid portion is a single-stranded DNA. The molecule according to claim 38 or 39, wherein the DNA contained in the nucleic acid portion is a double-stranded DNA. 43. The molecule according to any one of claims 38 to 42, wherein the DNA contained in the nucleic acid portion is generated by a reverse transcription reaction of RNA. Nucleoside or nucleoside-like chemical structural skeleton, in which the nucleic acid part can be covalently bonded to a substance having a chemical structural skeleton similar to an amino acid or an amino acid via a spacer on the 3′-terminal side of the base sequence reflecting the genotype 44. The molecule according to any one of claims 38 to 43, wherein the molecule has a substance having the following formula: The molecule according to claim 44, wherein the nucleoside or the substance having a chemical structure skeleton similar to the nucleoside is puromycin or a derivative thereof. 46. The molecule according to claim 44 or 45, wherein the spacer comprises a polymer. 47. The molecule according to any of claims 44 to 46, wherein the spacer has a length of 100-1000 °. 48. The molecule according to any of claims 44 to 47, wherein the spacer comprises a nucleic acid. The nucleic acid is a single strand of RNA or DNA, a double strand of RNA or DNA and DNA, a double strand of RNA and short PNA or DNA, a single strand of RNA and DNA, or 49. The molecule according to claim 48, which is a double strand of a single strand consisting of DNA and a short strand of DNA. 50. The molecule according to any of claims 44 to 49, wherein the spacer comprises polyethylene glycol. The molecule according to claim 50, wherein the spacer comprises polyethylene glycol and DNA. 52. The method according to claim 50 or 51, wherein the polyethylene glycol has a molecular weight of 3,000 to 30,000. 53. The molecule according to any one of claims 38 to 52, wherein the gene is an antibody or a partial gene thereof. 53. The molecule according to any one of claims 38 to 52, wherein the gene is an enzyme or a partial gene thereof. 53. The molecule according to any one of claims 38 to 52, wherein the gene comprises a random base sequence. The library according to any one of claims 38 to 54, comprising a plurality of molecules having different genes.

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