WO2000012530A1

WO2000012530A1 - dU SITE-DIRECTED CLEAVAGE OF COVALENT CONJUGATES

Info

Publication number: WO2000012530A1
Application number: PCT/US1999/018091
Authority: WO
Inventors: Russ Lehrman; Stephanie Niewlandt
Original assignee: Nexstar Pharmaceuticals Inc
Current assignee: Nexstar Pharmaceuticals Inc
Priority date: 1998-08-26
Filing date: 1999-08-10
Publication date: 2000-03-09
Anticipated expiration: 2001-02-26
Also published as: AU5346299A

Abstract

This invention discloses a method for identifying and preparing covalent conjugates comprising a nucleic acid component covalently bonded to a separable unit via a deoxyuridine. This invention further discloses covalent conjugates comprising a nucleic acid component covalently bonded to a separable unit via a deoxyuridine. This invention further discloses a method of analyzing a nucleic acid component of a covalent conjugate comprising a nucleic acid component covalently bonded to a separable unit via a deoxyuridine.

Description

dU Site-Directed Cleavage of Covalent Conjugates

FIELD OF THE INVENTION

This invention relates to covalent conjugates and prodrug forms of nucleic acids and their use in the purification, pharmaceutical and diagnostic arts. Specifically, this invention

describes a method for identifying covalent conjugates comprising a nucleic acid component covalently bonded to a separable unit via a deoxyuridine. This invention further describes a method of analyzing the nucleic acid component of a covalent conjugate by treating the

covalent conjugate with uracil DNA glycosylase. This invention further describes the use of

the covalent conjugates of the present invention as therapeutic and diagnostic agents.

BACKGROUND

Until quite recently, the consideration of oligonucleotides in any capacity other than strictly informational was unheard of. Despite the fact that certain oligonucleotides were known to have interesting structural possibilities (e.g., t-RNAs) and other oligonucleotides

were bound specifically by polypeptides in nature, very little attention had been focused on the noninformational capacities of oligonucleotides. For this reason, among others, little

consideration had been given to using oligonucleotides as pharmaceutical compounds.

There are currently several areas of exploration that have led to extensive studies regarding the use of oligonucleotides as pharmaceutical compounds. Antisense

oligonucleotides are used to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions. Further, the discovery of the

SELEX process (Systematic Evolution of Ligands by Exponential Enrichment (Tuerk and Gold (1990) Science 249:505; United States Patent No. 5,475,096; United States Patent No. 5,270,163) has shown that oligonucleotides can be identified that will bind to almost any biologically interesting target. The SELEX Process

The dogma for many years was that nucleic acids had primarily an informational

role. Through a method known as Systematic Evolution of Ligands by Exponential

Enrichment, termed SELEX, it has become clear that nucleic acids have three dimensional

structural diversity not unlike proteins. SELEX is a method for the in vitro evolution of

nucleic acid molecules with highly specific binding to target molecules and is described in

United States Patent Application Serial No. 07/536,428, filed June 11, 1990, entitled

"Systematic Evolution of Ligands by Exponential Enrichment," now abandoned; United

States Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled "Nucleic Acid

Ligands," now United States Patent No. 5,475,096; United States Patent Application Serial

No. 07/931,473, filed August 17, 1992, entitled "Methods for Identifying Nucleic Acid

Ligands," now United States Patent No. 5,270,163 (see also WO 91/19813), each of which

is specifically incorporated by reference herein. Each of these applications, collectively

referred to herein as the SELEX Patent Applications, describes a fundamentally novel

method for making a nucleic acid ligand to any desired target molecule. The SELEX

process provides a class of products which are referred to as nucleic acid ligands (also

referred to in the art as "aptamers"), each ligand having a unique sequence, and which has

the property of binding specifically to a desired target compound or molecule. Each

SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or

molecule. SELEX is based on the unique insight that nucleic acids have sufficient capacity

for forming a variety of two- and three-dimensional structures and sufficient chemical

versatility available within their monomers to act as ligands (form specific binding pairs)

with virtually any chemical compound. Molecules of any size or composition can serve as

targets. The SELEX method involves selection from a mixture of candidate oligonucleotides

and step- wise iterations of binding, partitioning and amplification, using the same general

selection scheme, to achieve virtually any desired criterion of binding affinity and

selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of

randomized sequence, the SELEX method includes steps of contacting the mixture with the

target under conditions favorable for binding, partitioning unbound nucleic acids from those

nucleic acids which have bound specifically to target molecules, dissociating the nucleic

acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target

complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of

binding, partitioning, dissociating and amplifying through as many cycles as desired to yield

highly specific high affinity nucleic acid ligands to the target molecule.

The SELEX method demonstrates that nucleic acids as chemical compounds can

form a wide array of shapes, sizes and configurations, and are capable of a far broader

repertoire of binding and other functions than those displayed by nucleic acids in biological

systems. The present inventors have recognized that SELEX or SELEX-like processes

could also be used to identify nucleic acids which can facilitate any chosen reaction in a

manner similar to that in which nucleic acid ligands can be identified for any given target.

In theory, within a candidate mixture of approximately 10¹³ to 10¹⁸ nucleic acids, the present

inventors postulate that at least one nucleic acid exists with the appropriate shape to

facilitate each of a broad variety of physical and chemical interactions.

The basic SELEX method has been modified to achieve a number of specific

objectives. For example, United States Patent Application Serial No. 07/960,093, filed

October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure,"

now abandoned (see also United States Patent No. 5,707,796), describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific

structural characteristics, such as bent DNA. United States Patent Application Serial No.

08/123,935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands,"

now abandoned (see also United States Patent Application No. 5,763,177), describes a

SELEX based method for selecting nucleic acid ligands containing photoreactive groups

capable of binding and/or photocrosslinking and/or photoinactiviating a target molecule.

United States Patent Application Serial No. 08/134,028, filed October 7, 1993, entitled

"High- Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and

Caffeine," now abandoned (see also United States Patent No. 5,580,737), describes a

method for identifying highly specific nucleic acid ligands able to discriminate between

closely related molecules, which can be non-peptidic, termed Counter-SELEX. United

States Patent Application Serial No. 08/143,564, filed October 25, 1993, entitled

"Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX," now

abandoned (see also United States Patent No. 5,567,588), describes a SELEX-based method

which achieves highly efficient partitioning between oligonucleotides having high and low

affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleic acid

ligands containing modified nucleotides conferring improved characteristics on the ligand,

such as improved in vivo stability or improved delivery characteristics. Examples of such

modifications include chemical substitutions at the ribose and/or phosphate and/or base

positions. SELEX-identified nucleic acid ligands containing modified nucleotides are

described in United States Patent Application Serial No. 08/117,991, filed September 8,

1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," now

abandoned (see also United States Patent No. 5,660,985), that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of

pyrimidines. United States Patent Application Serial No. 08/134,028, supra, describes

highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-

amino (2'-NH₂), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States Patent

Application Serial No. 08/264,029, filed June 22, 1994, entitled "Novel Method of

Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic

Displacement," now abandoned, describes oligonucleotides containing various 2'-modified

pyrimidines.

The SELEX method encompasses combining selected oligonucleotides with other

selected oligonucleotides and non-oligonucleotide functional units as described in United

States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled "Systematic

Evolution of Ligands by Exponential Enrichment: Chimeric SELEX," now United States

Patent No. 5,637,459, and United States Patent Application Serial No. 08/234,997, filed

April 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment:

Blended SELEX," now United States Patent No. 5,683,867, respectively. These

applications allow the combination of the broad array of shapes and other properties, and the

efficient amplification and replication properties, of oligonucleotides with the desirable

properties of other molecules.

The SELEX method encompasses complexes of oligonucleotides. United States

Patent Application Serial No. 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand

Complexes," describes a method for preparing a therapeutic or diagnostic complex

comprised of a nucleic acid ligand and a lipophilic compound or a non-immunogenic, high

molecular weight compound. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are

specifically incorporated by reference herein in their entirety.

Covalent Conjugates

It is widely accepted that the usefulness of many therapeutic agents is dependent on

their ability to be taken up by their target cell population. One approach for effecting

targeted drug delivery has been to use antisense oligonucleotides that are complementary

and can hybridize to selected cellular or viral target nucleic acid sequences to modulate the

expression of the target nucleic acid sequence. Antisense oligonucleotides, therefore, are

only effective as intracellular agents. However, the poor absorption of unmodified

oligomers by cells and their sensitivity to cellular nucleases and nucleases present in culture

medium and serum limits their use both in vivo and in vitro. An additional approach for

effecting drug delivery to a target site involves forming a covalent conjugate by attaching

the drug to a carrier capable of transporting and/or targeting the drug from the site of

application directly to the site of action. Such carriers may be divided into four categories:

(1) linear polymers; (2) cells; (3) three dimensional systems (e.g., liposomes); and (4)

ligands that recognize specific cellular components (e.g., cell-surface receptors). Once the

conjugate is in the target area, the linkage between the drug and the carrier is cleaved

effecting the release of the drug. Examples of such carriers include proteins, which are

cleaved by proteolytic enzymes inside the cell, polysaccharides which are cleaved by

glycosidases, or vinyl polymers which contain hydrolytically labile ester bonds.

Conjugating an oligonucleotide, for example, to a cholesterol will increase the efficiency of

uptake of the oligonucleotide by the cell (Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA

86:6553-6556). In PCT Application International Publication No. WO 91/14696, Latham et

al. describe a method for chemically modifying antisense oligonucleotides to enhance entry of the drug into a cell by conjugating an oligonucleotide via a disulfide bond to a transport

agent. Biopolymers, therefore can be constructed to possess domains that have

complementary or independent functions in cassette form in which specific domains are

linked together to aid in the construction of clinically useful molecules. Non-natural

hybrids have been prepared in which DNA is linked to targeting, cleaving or reporter groups

(Zuckermann et al. (1987) Nucleic Acid Research 15:5305).

A few instances have been reported where researchers have attached antisense

oligonucleotides to lipophilic compounds or non-immunogenic, high molecular weight

compounds (See, e.g., United States Patent Application Serial No. 08/434,465, supra). A

lipophilic compound covalently attached to an antisense oligonucleotide through a

phosphoester bond has been described in EP 462 145 Bl of Bischofberger.

Linear polymers are typically covalently linked to a drug via a hydrolyzable bond

such as an ester (Hoes and Feijen (1989) Drug Carrier Systems, eds. F.H.D. Roerdink and

A.M. Kroon, John Wiley & Sons, N.Y., pp. 57-109). Base hydrolysis of these compounds

cleaves the ester linkage and permits the characterization of the drug free of the linear

polymer. However, since the ester cleavage must be performed at elevated pH (>10), the

process is limited to compounds that are not otherwise base sensitive. For example,

covalent conjugates comprising an RNA component cannot be treated at elevated pH

because the phosphodiester backbone of the RNA is readily hydrolyzed under such

conditions. In addition, ester bonds can be quickly hydrolyzed in biological fluids by

extracellular enzymes which cleave ester bonds. Therefore, the half life in vivo of a nucleic

acid conjugated to, for example, a lipophilic compound through an ester bond may be too

short to permit a therapeutic response. Furthermore, the ester linkage can increase the instability of such compounds due to hydrolysis of the ester bond, which can occur during

normal storage conditions.

Uracil DNA Glvcosylase

DNA glycosylases initiate DNA repair pathways in many organisms by excising

unconventional or damaged bases from DNA. Uracil DNA glycosylases (UDG), purified

from E. Coli and several other sources, are concentrated in the cellular nuclei and are found

to be the most abundant of all the glycosylases in the cell. UDG eliminates uracil from

DNA containing deoxyuridine (dU) by cleaving the N-glycosidic bond between the base

and the sugar-phosphate backbone. The resulting apyrimidine is readily cleaved at neutral

pH at elevated temperatures or under mildly basic conditions. UDG does not remove uracil

from RNA or free nucleotides, but only from DNA (single- or double-stranded) longer than

about four nucleotides. (Duncan (1981) The Enzymes. 3rd ed., part A, Academic Press,

N.Y., pp. 565-586).

Intentional dU incorporation into oligonucleotides has been described for

biochemical and molecular biological applications. One example of the use of UDG has

been applied to polymerase chain reactions (PCRs). The PCR procedure amplifies specific

nucleic acid sequences through a series of manipulations, resulting in the synthesis of

abundant amplification products. Contamination of new PCRs with trace amounts of these

products, called carry-over contamination, yields false positive results. It has been found

that carry-over contamination in PCRs can be controlled by incorporating dUTP in all PCR

products and then treating all subsequent fully preassembled starting reactions with UDG,

followed by thermal inactivation of UDG (Longo et al. (1990) Gene 93:125-128; Hartley,

United States Patent No. 5,035,996). A consequence of this method is that the high

frequency of dU incorporation within any given sequence guarantees that treatment with UDG produces a complex mixture of oligonucleotide fragments which can be difficult to

analyze. dU has also been used to reduce background in site-directed mutagenesis (Horwitz

and DiMaio (1990) Methods in Enzymology 185:599-611; Kunkel (1985) Prod. Natl. Acad.

Sci., USA 82:488-492).

Since enzymes which cleave DNA do not have high specificity for the cleavage of

single stranded DNA, site-directed cleavage of DNA is most often performed using metal

complexes (Sigman et al. (1993) Chemical Reviews 93:2295-2316; Pyle and Barton (1990)

Progress in Inorganic Chemistry: Bioinorganic Chemistry, ed. S.J. Lippard, vol. 38, pp.

413-475; Muller et al. (1996) J. Amer. Chem. Soc. 118:2320-2325). In the case of RNA,

nucleases are known which specifically cleave single-stranded RNA at individual

nucleotides (Zuckermann and Schultz (1988) J. Am. Chem. Soc. 110:6592-6594), but the

redundancy of cleavage ensures that in almost all situations nuclease treatment will result in

multiple fragments. Type II restriction endonucleases have been well characterized. One

example is Fokl which is known to specifically recognize a pentanucleotide sequence in

unmodified double-stranded DNA and cuts at a defined distance from the right-hand side of

the recognition sequence (Eun (1996) Enzymology Primer for Recombinant DNA

Technology, Academic Press, pp. 267-276).

SUMMARY OF THE INVENTION

The present invention provides covalent conjugates comprising one or more nucleic

acid components covalently conjugated to a separable unit through one or more

deoxyuridine residues.

In one embodiment the nucleic acid component of the covalent conjugate is a nucleic

acid ligand. In the preferred embodiment, the nucleic acid ligand is identified according to the SELEX method. In one embodiment, the nucleic acid component is single-stranded

DNA. In another embodiment, the nucleic acid component is RNA. In embodiments in

which the RNA nucleic acid component is a nucleic acid wherein all nucleotides are 2'-OH

nucleotides, the covalent conjugate contains one or more DNA residues between the dU and

the RNA nucleic acid component.

In one embodiment of the invention, the separable unit is a nucleic acid. In another

embodiment of the invention the separable unit is a non-immunogenic, high molecular

weight compound. In a preferred embodiment, the non-immunogenic, high molecular

weight compound is polyethylene glycol (PEG). In another embodiment of the invention,

the separable unit is a lipophilic compound. In a preferred embodiment, the lipophilic

compound is cholesterol, a dialkyl glycerol or a diacyl glycerol.

In another embodiment, the present invention provides means for the site directed

cleavage of a covalent conjugate comprising a nucleic acid and a separable unit by

incorporating a non-hydrolyzable linker, a deoxyuridine (dU), at the junction between the

nucleic acid and the separable unit. When treated with UDG followed by mildly basic

conditions, the nucleic acid is site-specifically cleaved from the attached separable unit

without effecting the oligonucleotide

In another embodiment, this invention provides a method for targeting a therapeutic

or diagnostic agent to a specific predetermined target in a patient comprising administering

a covalent conjugate to the patient. In one embodiment, the therapeutic or diagnostic agent

is the nucleic acid component of the covalent conjugate. In one embodiment, the covalent

conjugate comprises a nucleic acid component which is inactive when linked to the

separable unit and is activated when cleaved from the separable unit after administration of

an appropriate enzyme. The present invention further provides a method for analyzing the structure of a

nucleic acid component of a covalent conjugate.

The present invention further provides means for analyzing a nucleic acid

component of a covalent conjugate of the present invention after the covalent conjugate has

been administered to a patient, comprising administering the covalent conjugate to a patient,

isolating the covalent conjugate from a biological fluid after a period of time and cleaving

the nucleic acid from the separable unit.

This invention further provides covalent conjugates wherein the nucleic acid

component of the covalent conjugate is an intracellular agent and the separable unit is

capable of transporting the nucleic acid into a cell. Once the covalent conjugate is in the

target cell, the separable unit is cleaved by the intracellular UDG, effecting the release of the

nucleic acid.

These and other objects, as well as the nature, scope and utilization of this invention,

will become readily apparent to those skilled in the art from the following description and

the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the anion exchange HPLC trace of the digestion of 5K-PEG-dU303

(SEQ ID NO:l) with UDG in UDG Buffer pH 8.4 at 90°C for 1 hour. The peak at 16.2

minutes is the 5K-PEG-dU303, and the peak at 19.1 min. is the digestion product.

Figure 2 shows the anion exchange HPLC trace of the digestion of

dTdUdTrArCrGrArCrGrA (SEQ ID NO:8) with UDG in UDG Buffer pH 7.8 (Tris) at 37°C

for 2 hours followed by 65°C for 5 hours. All chromatographic peaks observed before 10

minutes were also observed in a negative control without oligonucleotide present. Figure 3 shows the anion exchange HPLC trace of the digestion of

dUdTrArCrGrArCrGrA (SEQ ID NO:15)with UDG in UDG Buffer pH 7.8 (Tris) at 37°C

for 3 hours followed by 65°C for 20 hours. All chromatographic peaks observed before 10

minutes were also observed in a negative control without oligo present.

DETAILED DESCRIPTION OF THE INVENTION

acid components covalently conjugated to a separable unit through one or more

deoxyuridine residues. The covalent conjugates of the present invention have the formula

A-B-C, wherein A is RNA, single-stranded DNA or double stranded DNA, B is one or more

deoxyuridines and C is a separable unit, wherein both A and C are covalently bonded to B.

In embodiments wherein A is RNA comprising all 2'-OH nucleotides, a DNA residue is

incorporated between A and B.

In one embodiment of the invention, the nucleic acid component of the covalent

conjugate is single-stranded DNA. In another embodiment, the nucleic acid component is

RNA. In one embodiment, the nucleic acid component is a nucleic acid ligand. In another

embodiment, the nucleic acid component is a nucleic acid ligand identified by the SELEX

method. In one embodiment, the separable unit is bound to a hydroxyl group at the 5' or 3'

terminus of the nucleic acid component.

In embodiments where the nucleic acid is 2'-OH RNA the covalent conjugate

comprises at least one DNA residue incorporated between the dU and the 2'-OH RNA

nucleic acid component. It was observed that the rate of cleavage of the separable unit from

a covalent conjugate comprising a 2'-OH RNA as the nucleic acid component was much

slower compared to the rate of cleavage of the separable unit from covalent conjugates comprising DNA as the nucleic acid component. However, it was discovered that by

incorporating a DNA residue between the dU and the 2'-OH RNA nucleic acid component,

the rate of cleavage of the separable unit increased to a rate comparable the rate of cleavage

of covalent conjugates comprising DNA nucleic acid components. While not wishing to be

bound by any theory, it is believed that the slow rate of cleavage may be due to the presence

of the 2'-OH. Therefore, it is believed that the rate of cleavage of an RNA nucleic acid

component from a covalent conjugate will be significantly increased by incorporating a

number of 2'-modified pyrimidines (for example, 2'-F, 2'-NH₂ or 2'-OMe). The inventors

believe that these 2'-modifications will increase the rate of cleavage to the extent that it will

not be necessary to incorporate a DNA residue between the dU and the 2'-modified RNA

nucleic acid component.

In one embodiment of this invention, the separable unit of the covalent conjugate is

a lipophilic compound such as cholesterol, a dialkyl glycerol, a diacyl glycerol, or a non-

immunogenic, high molecular weight compound such as PEG. In these cases, the

pharmacokinetic properties of the covalent conjugate will be enhanced relative to the

nucleic acid alone.

In one embodiment, the separable unit is covalently attached to the dU. In another

embodiment, the separable unit is covalently attached to the dU through a linker. In

embodiments where the conjugate comprises a linker between the separable unit and the dU,

the linker comprises a reactive group. In one embodiment, the linker comprises a primary

amine.

Before proceeding further with the description of the specific embodiments of the

present invention, a number of terms will be defined. "Nucleic acid ligand" as used herein is a nucleic acid having a desirable action on a

target. A desirable action includes, but is not limited to, binding of the target, catalytically

changing the target, reacting with the target in a way which modifies/alters the target or the

functional activity of the target, covalently attaching to the target as in a suicide inhibitor or

facilitating a reaction between the target and another molecule. In the preferred

embodiment, the action is specific binding affinity for a target molecule, such target

molecule being a three dimensional chemical structure other than a polynucleoti.de that

binds to the nucleic acid ligand through a mechanism which predominantly depends on

Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a

nucleic acid having the known physiological function of being bound by the target

molecule. In one embodiment, the nucleic acid ligand is a non-naturally occurring nucleic

acid. In preferred embodiments of the invention, the nucleic acid ligands are identified by

the SELEX methodology. Nucleic acid ligands include nucleic acids that are identified

from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given

target, by the method comprising a) contacting the candidate mixture with the target,

wherein nucleic acids having an increased affinity to the target relative to the candidate

mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the

increased affinity nucleic acids from the remainder of the candidate mixture; and c)

amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic

acids.

"Candidate Mixture" is a mixture of nucleic acids of differing sequence from which

to select a desired ligand. The source of a candidate mixture can be from naturally-

occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids,

enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques. In a preferred embodiment, each nucleic acid has fixed sequences to

facilitate the amplification process surrounding a randomized region of sequences.

"Nucleic acid" means either DNA, RNA, single-stranded or double-stranded and any

chemical modifications thereof. Modifications include, but are not limited to, those which

provide other chemical groups that incorporate additional charge, polarizability, hydrogen

bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or the

nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-

position sugar modifications, 5-position pyrimidine modifications, 8-position purine

modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution

of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, unusual base-pairing

combinations such as the isobases isocytidine and isoguanidine and the like. Modifications

can also include 3' and 5' modifications such as capping.

"Non-immunogenic, high molecular weight compound" is a compound of

approximately 1000 Da or more that typically does not generate an immunogenic response.

An immunogenic response is one that induces the organism to produce antibody proteins.

Examples of non-immunogenic, high molecular weight compounds include polyethylene

glycol (PEG); polysaccharides, such as dextran; polypeptides, such as albumin; and

magnetic structures, such as magnetite. In certain embodiments, the non-immunogenic,

high molecular weight compound can also be a nucleic acid ligand.

"Lipophilic compounds" are compounds which have the propensity to associate with

or partition into lipid and/or other materials or phases with low dielectric constants,

including structures that are comprised substantially of lipophilic components. Cholesterol,

phospholipids, diacyl glycerols and dialkyl glycerols are examples of lipophilic compounds. "SELEX" methodology involves the combination of selection of nucleic acid ligands

which interact with a target in a desirable manner, for example binding to a protein, with

amplification of those selected nucleic acids. Iterative cycling of the selection/amplification

steps allows selection of one or a small number of nucleic acids which interact most

strongly with the target from a pool which contains a very large number of nucleic acids.

Cycling of the selection/amplification procedure is continued until a selected goal is

achieved. In the present invention the SELEX methodology can be employed to obtain a

nucleic acid ligand to a desirable target.

The SELEX methodology is described in the SELEX Patent Applications.

"Target" means any compound of interest for which a ligand is desired. A target

molecule can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone,

receptor, antigen, antibody, virus, substrate, metabolite, cell, tissue, transition state analog,

cofactor, inhibitor, drug, dye, nutrient, growth factor, etc., without limitation.

"Improved pharmacokinetic properties" means that the nucleic acid component

covalently bonded to the separable unit through a deoxyuridine shows a longer circulation

half-life in vivo relative to a nucleic acid not covalently bonded to the separable unit, or has

other pharmacokinetic benefits such as improved target to non-target concentration ratio.

"Covalent conjugate" as used herein describes the molecular entity having the

formula A-B-C, wherein A is RNA, single-stranded DNA or double stranded DNA, B is one

or more deoxyuridines and C is a separable unit, wherein both A and C are covalently

bonded to B. In embodiments wherein A is RNA comprising all 2'-OH nucleotides, a DNA

residue is incorporated between A and B.

"Separable unit" as used herein includes oligonucleotides, lipophilic compounds,

non-immunogenic, high molecular weight compounds, nucleic acids, proteins, carbohydrates, organic compounds that chelate metals, detectable moieties such as

fluorescent, chemiluminescent and bioluminescent marker compounds, antibodies, biotin,

etc.

"Uracil DNA glycosylase" (UDG), a term of art, is a DNA glycosylase which

excises damaged or unconventional bases from DNA and initiates the DNA repair pathway.

UDG eliminates uracil from DNA by cleaving the N-glycosidic bond between the base

uracil and the deoxyribose-phosphate backbone, only when the monomeric nucleotide dUTP

is incorporated into a DNA molecule, resulting in incorporation of deoxyuridine. UDG

does not remove uracil from not free dUTP, free deoxyuridine or RNA. (Duncan (1981) in

The Enzymes, ed. P. Boyer, vol. XIV, pp. 565-586.)

"Deoxyuridine" is a nucleoside residue which can arise as a result of either

misincorporation of deoxyuridine monophosphate residues by DNA polymerase or due to

deamination of cytosine.

"Linker" is a molecular entity that connects two or more molecular entities through a

covalent bond. Examples of linkers include an organic compound that can be attached to an

oligonucleotide and has a chemically reactive group such as a free amino, sulfhydryl, diene,

dienophile etc.

"Covalent bond" is the chemical bond formed by the sharing of electrons.

"Therapeutic Agent" means a compound which is used in the treatment of diseases

and disorders. In some applications of the invention, the nucleic acid component of the

covalent conjugate can be for targeting purposes and the separable unit is the therapeutic

agent. In other applications, the nucleic acid component of the covalent conjugate is the

therapeutic agent. "Diagnostic Agent" means a covalent conjugate which can be used for detecting the

presence or absence of and/or measuring the amount of target in a sample. Detection of the

target molecule is mediated by its binding to a nucleic acid component of a covalent

conjugate specific for that target molecule. The covalent conjugate can be labeled, for

example radiolabeled, to allow qualitative or quantitative detection.

acid components covalently bonded to a separable unit through one or more deoxyuridines.

Such complexes have the following advantages over nucleic acids covalently bonded to a

separable unit through a chemical unit other than a dU. First, incorporating a dU at a

specific location in a covalent conjugate, wherein the dU is at the junction between the

nucleic acid component and the separable unit, provides means for site-directed cleavage

specifically at the dU by treating the covalent conjugate with an appropriate enzyme, such

as UDG under mild conditions wherein the nucleic acid remains intact. Second, linking the

nucleic acid component to a separable unit through a dU imparts improved stability to the

covalent conjugate relative to covalent conjugates comprising hydrolyzable ester linkages,

since the dU linkage is stable to hydrolysis prior to enzymatic cleavage of the base. Third,

use of a dU linkage between a nucleic acid component and a separable unit results in

improved pharmacokinetic properties (e.g., longer half-life) of the covalent conjugate in-

vivo relative to the nucleic acids alone, since UDG is concentrated in cellular nuclei. For

example, the presence of dU in the covalent conjugate confers stability under extracellular

conditions. However, under intracellular conditions the covalent conjugate is cleaved by

intracellular UDG. And forth, since the cleavage of the nucleic acid from the separable unit

occurs at a pH lower than that used for cleavage of covalent conjugates containing an ester

linkage, the present invention allows for the utilization of base-sensitive RNA as the nucleic acid component of the covalent conjugate. Therefore, the separable unit enhances certain

properties of the covalent conjugate relative to the nucleic acid alone, while the dU allows

for a method of site-directed cleavage of the nucleic acid component from the separable unit

in such a way that the nucleic acid remains intact.

The present invention provides methods for the isolation of the nucleic acid cleaved

from a covalent conjugate for further analysis. For example, typically in a covalent

conjugate comprising a nucleic acid component coupled to a PEG, the nucleic acid is

coupled to a PEG as the final step in the synthesis of the nucleic acid to form the covalent

conjugate, and then the covalent conjugate product is purified, usually by extraction into an

organic solvent. Thus, the final nucleic acid product is not characterized prior to

PEGylation. The ability to specifically cleave the nucleic acid component covalently

coupled to a PEG through a dU by the method of the invention, in a manner which does not

degrade the nucleic acid, allows one to accurately verify the structure of the final nucleic

acid following purification. Thus, in the method of the present invention, a nucleic acid can

be linked to a separable unit such as a PEG through a deoxyuridine prior to purification to

form a covalent conjugate. The covalent conjugate can then be easily purified, and

following purification the nucleic acid component of the covalent conjugate can be cleaved

from the separable unit by treatment with an appropriate enzyme such as UDG, and the

nucleic acid can be analyzed.

The method of the present invention further provides covalent conjugates

comprising an active nucleic acid which is inactive while conjugated to the separable unit.

In this embodiment, the covalent conjugate is administered to a patient and the nucleic acid

is activated at a specific time by administration of an appropriate enzyme such as UDG to

cleave the active nucleic acid from the separable unit. In another embodiment, the nucleic acid component of a covalent conjugate is active

when in a hairpin or stem-loop or pseudoknot conformation, and the separable unit is a

nucleic acid sequence which is complementary to a portion of the nucleic acid and thus

inactivates the nucleic acid component by disrupting the active conformation. Site specific

cleavage of the covalent conjugate by the addition of UDG decreases the strength of the

interaction between the separable unit and the nucleic acid component, and the nucleic acid

component then returns to the active conformation.

The covalent conjugates of the present invention may be used as therapeutic or

diagnostic agents. Thus the present invention provides means for treating a disease using

covalent conjugates of the present invention. In one embodiment where the covalent

conjugate is administered to a patient for diagnostic or therapeutic purposes, the covalent

conjugate comprises a nucleic acid component which is inactive while covalently bonded to

the separable unit through a dU. The covalent conjugate remains intact until administration

of UDG which effects cleavage of the nucleic acid component from the separable unit. In

another embodiment, the nucleic acid component of the covalent conjugate is an

intracellular agent, wherein the separable unit is a carrier capable of transporting the nucleic

acid component into a target cell. Once the covalent conjugate is in the target cell, the

nucleic acid component activates upon cleavage from the covalent conjugate by the

intracellular enzyme UDG.

If a covalent conjugate is to be used as a therapeutic agent, it may be desirable to

analyze the composition of the covalent conjugate after it has been administered to a patient.

The ability to cleave a covalent conjugate at a specific site facilitates accurate detailed

structural characterization of covalent conjugate which has been exposed to biofluids for

pharmacokinetic or drug metabolism studies. The covalent conjugates described herein are particularly useful as in vivo or in vitro

diagnostics. In one embodiment, the covalent conjugates of the present invention are used

as diagnostic agents for detecting the presence or absence of a target molecule in a sample.

In one embodiment, the covalent conjugate comprises a nucleic acid ligand to a desired

target, and the separable unit comprises a detectable moiety such as a biotin, an enzyme or a

fluorophore. Various means of detection using such labels are known in the art and when

they specifically bind to or interact with a target site, the binding or interaction can be

observed by detecting the label. This method can be useful as a diagnostic tool, for example

to determine whether a particular target is present in a sample by adding a covalent

conjugate comprising a specific oligonucleotide that selectively binds to or interacts with

the target, washing away unbound covalent conjugate, and observing binding or interaction

by looking for the label.

One problem encountered in the therapeutic and in vivo diagnostic use of nucleic

acids is that oligonucleotides in the phosphodiester form can be quickly degraded in body

fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases

before the desired effect is manifest. Certain chemical modifications of the nucleic acid

ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance

or to mediate the delivery of the nucleic acid ligand. Modification of the nucleic acid

ligands contemplated in this invention include, but are not limited to, those which provide

other chemical groups that incorporate additional charge, polarizability, hydrophobicity,

hydrogen bonding, electrostatic interaction, steric bulk and fluxionality to the nucleic acid

ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are

not limited to 2'-position sugar modifications, 5-position pyrimidine modifications, 8-

position purine modifications, modifications at exocyclic amines, substitution of 4- thiouridine, substitution of 5-bromo or 5-iodouracil; backbone modifications,

phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing

can also include 3' and 5' modifications such as capping.

Where the nucleic acid ligands are derived by the SELEX method, the modifications

can be pre- or post- SELEX modifications. Pre-SELEX modifications yield nucleic acid

ligands with both specificity for their SELEX target and improved in vivo stability without

adversely affecting the binding capacity of the nucleic acid ligands. The preferred

modifications of the nucleic acid ligands of the invention are 5' and 3' phosphorothioate

capping or 3'-3' inverted phosphodiester linkage at the 3' end. For RNA ligands, additional

2' modifications, including 2'-F, 2'-OMe, and 2'-NH₂ modifications of some or all of the

nucleotides is preferred.

In the preferred embodiment, the nucleic acid ligands of the present invention are

derived from the SELEX methodology. SELEX is described in United States Patent

Application Serial No. 07/536,428, entitled "Systematic Evolution of Ligands by

Exponential Enrichment," now abandoned, United States Patent Application Serial No.

07/714,131, filed June 10, 1991, entitled "Nucleic Acid Ligands," now United States Patent

No. 5,475,096 and United States Patent Application Serial No. 07/931,473, filed August 17,

1992, entitled "Methods for Identifying Nucleic Acid Ligands," now United States Patent

No. 5,270,163 (see also WO91/19813). These applications, each specifically incorporated

herein by reference, are collectively called the SELEX Patent Applications.

The SELEX process provides a class of products which are nucleic acid molecules,

each having a unique sequence, and each of which has the property of binding specifically

to a desired target compound or molecule. Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules.

SELEX methodology can also be used to target biological structures, such as cell surfaces or

viruses, through specific interaction with a molecule that is an integral part of that biological

structure.

In its most basic form, the SELEX process may be defined by the following series of

steps:

1) A candidate mixture of nucleic acids of differing sequence is prepared. The

candidate mixture generally includes regions of fixed sequences (i.e., each of the members

of the candidate mixture contains the same sequences in the same location) and regions of

randomized sequences. The fixed sequence regions are selected either: (a) to assist in the

amplification steps described below, (b) to mimic a sequence known to bind to the target, or

(c) to enhance the concentration of a given structural arrangement of the nucleic acids in the

candidate mixture. The randomized sequences can be totally randomized (i.e., the

probability of finding a base at any position being one in four) or only partially randomized

(e.g., the probability of finding a base at any location can be selected at any level between 0

and 100 percent).

2) The candidate mixture is contacted with the selected target under conditions

favorable for binding between the target and members of the candidate mixture. Under

these circumstances, the interaction between the target and the nucleic acids of the candidate

mixture can be considered as forming nucleic acid-target pairs between the target and those

nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target are partitioned from

those nucleic acids with lesser affinity to the target. Because only an extremely small

number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set

the partitioning criteria so that a significant amount of the nucleic acids in the candidate

mixture (approximately 5-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher

affinity for the target are then amplified to create a new candidate mixture that is enriched in

nucleic acids having a relatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newly formed

candidate mixture contains fewer and fewer unique sequences, and the average degree of

affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the

SELEX process will yield a candidate mixture containing one or a small number of unique

nucleic acids representing those nucleic acids from the original candidate mixture having

the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process in great

detail. Included are targets that can be used in the process; methods for the preparation of

the initial candidate mixture; methods for partitioning nucleic acids within a candidate

mixture; and methods for amplifying partitioned nucleic acids to generate enriched

candidate mixtures. The SELEX Patent Applications also describe ligand solutions

obtained to a number of target species, including both protein targets wherein the protein is

and is not a nucleic acid binding protein.

The basic SELEX method has been modified to achieve a number of specific

now abandoned (see also United States Patent No. 5,707,796), describes the use of SELEX

in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. United States Patent Application Serial No.

now abandoned (see United States Patent No. 5,763,177), describes a SELEX based method

for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or

photocrosslinking to and/or photoinactivating a target molecule. United States Patent

Application Serial No. 08/134,028, filed October 7, 1993, entitled "High-Affinity Nucleic

Acid Ligands That Discriminate Between Theophylline and Caffeine," now abandoned (see

also United States Patent No. 5,580,737), describes a method for identifying highly specific

nucleic acid ligands able to discriminate between closely related molecules, termed

Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25,

1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution

SELEX," now abandoned (see also United States Patent No. 5,567,588), describes a

SELEX-based method which achieves highly efficient partitioning between

oligonucleotides having high and low affinity for a target molecule. United States Patent

Application Serial No. 07/964,624, filed October 21, 1992, now issued as United States

Patent No. 5,496,938, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,"

describes methods for obtaining improved nucleic acid ligands after SELEX has been

performed. United States Patent Application Serial No. 08/400,440, filed March 8, 1995,

entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,"

now United States Patent No. 5,705,337, describes methods for covalently linking a ligand

to its target.

The SELEX method encompasses the identification of high-affinity nucleic acid

such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base

abandoned (see also United States Patent No. 5,660,985), that describes oligonucleotides

containing nucleotide derivatives chemically modified at the 5- and 2'-positions of

amino (2'- NH₂), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States Patent

pyrimidines.

The SELEX method encompasses combining selected oligonucleotides with other

Patent No. 5,637,459 and United States Patent Application Serial No. 08/234,997, filed

Blended SELEX," now United States Patent No. 5,683,867, respectively. These

properties of other molecules. United States Patent Application No. 08/434,465, filed May

4, 1995, entitled "Nucleic Acid Ligand Complexes," describes a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic

compound or a non-immunogenic, high molecular weight compound. Each of the above

described patent applications which describe modifications of the basic SELEX procedure

are specifically incorporated by reference herein in their entirety.

SELEX identifies nucleic acid ligands that are able to bind targets with high affinity

and with outstanding specificity, which represents a singular achievement that is

unprecedented in the field of nucleic acids research. These characteristics are, of course, the

desired properties one skilled in the art would seek in a therapeutic or diagnostic ligand.

In order to produce nucleic acid ligands desirable for use as a pharmaceutical, it is

preferred that the nucleic acid ligand (1) binds to the target in a manner capable of achieving

the desired effect on the target; (2) be as small as possible to obtain the desired effect; (3) be

as stable as possible; and (4) be a specific ligand to the chosen target. In most situations, it

is preferred that the nucleic acid ligand has the highest possible affinity to the target.

As described above, the covalent conjugates described herein are useful as

pharmaceuticals. Therapeutic compositions of the covalent conjugates may be administered

parenterally by injection, although other effective administration forms, such as

intraarticular injection, inhalant mists, orally active formulations, ointments, transdermal

iontophoresis or suppositories, are also envisioned. One preferred carrier is physiological

saline solution, but it is contemplated that other pharmaceutically acceptable carriers may

also be used. In one preferred embodiment, it is envisioned that the carrier and the ligand

constitute a physiologically-compatible, slow release formulation. The primary solvent in

such a carrier may be either aqueous or non-aqueous in nature. In addition the carrier may

contain other pharmacologically-acceptable excipients for modifying or maintaining the pH,

osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmacologically-acceptable

excipients for modifying or maintaining the stability, rate of dissolution, release, or

absorption of the ligand. Such excipients are those substances usually and customarily

employed to formulate dosages for parental administration in either unit dose or multi-dose

form.

Once the therapeutic composition has been formulated, it may be stored in sterile

vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.

Such formulations may be stored either in a ready to use form or requiring reconstitution

immediately prior to administration. The manner of administering formulations containing

covalent conjugates for systemic delivery may be via subcutaneous, intramuscular,

intravenous, intranasal or vaginal or rectal suppository. The manner of administering UDG

if it is desired to cleave the nucleic acid component from the covalent conjugate in vivo,

UDG may be via subcutaneous, intramuscular or intravenous injection.

Oligonucleotides containing dU were synthesized using standard phosphoramidite

chemistry on solid support and deprotected prior to use by standard methods. Polyethylene

glycol was conjugated to several oligonucleotides via an amine linker and the resulting

conjugated oligonucleotides were purified by organic extraction and liquid chromatographic

methods.

The following examples are provided to explain and illustrate the present invention

and are not to be taken as limiting of the invention. Example 1 describes the conjugation of

an oligodeoxyribonucleotide incorporating a single dU with a Methoxy-SPA 5,000 MW

polyethylene glycol, and describes the digestion of this product with UDG. The only

cleaved product of this digestion is 5'-phosphate-dAdCdTdGdAdCdGdA. Example 2

describes the conjugation of NX dU303 with a Methoxy-SPA-5,000 MW polyethylene glycol and the digestion of this product with UDG at 90°C. NX303 is an L-Selectin ligand

obtained by the method described in PCT Application International Publication No. WO

96/40703, published December 19, 1996, which is incorporated herein by reference. This

digestion resulted in clean cleavage of the PEG-5-amino-C6-dT-dU unit from the covalent

conjugate to give the product 5'-phosphate-

dCdCdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAd

AdTdGdG3'3'dTdT-3' (SEQ ID NO:4).

Example 3 was performed to investigate the reactivity of UDG on an oligonucleotide

containing 2'-fluorouridines and 2'-fluorocytosines. The purpose of this experiment was to

determine if UDG would recognize a 2'-fluoro uridine as a substrate for cleavage. NX1255

(SEQ ID NO:5), which contains a number of 2'-fluoro-modified nucleotides, but does not

contain a dU, was used in this experiment. This Example shows that UDG does not cleave

oligonucleotides containing 2'-fluoro-modified nucleosides. Only a minor amount of

normal degradation of the oligonucleotide was observed. Example 4 describes the digestion

with UDG of an RNA oligonucleotide containing a single dU. This digestion proceeded

much slower than the digestion of the DNA oligonucleotides.

Example 5 describes the digestion with UDG of an RNA oligonucleotide having a

dTdUdT at the 5' end of the oligonucleotide. This example demonstrates that incorporating

a single DNA nucleoside between the dU and an RNA oligonucleotide unmodified at the 2'-

position significantly increases the rate of digestion and the yield of cleanly cleaved

product. Example 6 describes the digestion with UDG of an RNA oligonucleotide having a

dUdT at the 5' end of the oligonucleotide. This example demonstrates that it is not

necessary to have the dU flanked by DNA nucleosides. Example 7 describes the digestion with UDG of an L-selectin specific nucleic acid

ligand 21918 (NH₂-C6-dUdTrGrGrArGfUfCfUfUrArGrGfCrArGfCrGfCrGfUfUfUfUfCr

GrArGfCfUrAfCfUfCfC3'3'dT (SEQ ID NO: 12). This oligonucleotide has been adapted

from an original nucleic acid ligand (NX 11702) by the inclusion of the dUdT next to the

C6-NH₂ linker. NX 11702 is described in PCT Application WO 96/40703 , which is

incorporated herein by reference. In addition to UDG digestion, the 21918 material was

subjected to a xenogenic lymphocyte trafficking experiment in which the results were

comparable to those obtained for the NX 11702 nucleic acid ligand. This example

demonstrates that inclusion of dUdT in the nucleic acid ligand sequence at the 5' end does

not negatively affect the inhibition properties of the nucleic acid ligand in an in vivo efficacy

indicating assay.

Example 8 describes the digestion with UDG of an anti-VEGF nucleic acid ligand

covalently linked to a 40K PEG (NX22022) (40K-PEG-C6-NH₂dT-dUdTfCmGmGrArA

fUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmGS^'dT) (SEQ ID

NO: 14). This oligonucleotide has been modified from an original nucleic acid ligand

(NX31838) by the inclusion of the dUdT. NX31838 is described in United States Patent

Application No. 08/870,930, filed June 6, 1997, entitled "Vascular Endothelial Growth

Factor (VEGF) Nucleic Acid Ligand Complexes." In addition to UDG digestion, NX22022

was subjected to a dermal vascular permeability assay in which the results were comparable

to those obtained for the NX31838 ligand. This example demonstrates that the inclusion of

dUdT in the ligand sequence towards the 5' end does not negatively affect the ability of the

ligand to attenuate VEGF-induced changes in the permeability of the dermal vasculature.

To evaluate the in vivo stability of the NX 22022 material containing dUdT, a

pharmacokinetic study was performed in rats with NX 22022 and NX31838. The major difference observed between NX22022 and NX31838 was the biphasic clearance of

NX22022 from plasma compared with the monoexponential clearance of NX31838. A

significant amount (75%) of NX22022 was cleared in the initial alpha phase with a t_1/2 of 25

minutes, resulting in a three fold faster clearance rate than NX31838. Conversely, the beta

phase t_1/2 (283 minutes) for the dU-containing ligand was found to be similar to the overall

t_1/2 (351 minutes) of the standard NX31838 ligand. These date suggest that the dU linkage

is recognized by an endogenous nuclease in rats.

EXAMPLES

The following materials and methods were used throughout.

A. Materials

Uracil DNA Glycosylase was obtained from Giboco Life Technologies (Cat. No.

18054-015) at lunit/μL in 30 niM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM

DTT, 0.05% (w/v) Tween 20, 50% (w/v) glycerol.

UDG Buffers. All buffers were prepared using fresh solutions and filtered with 0.45 μm

nylon filter prior to use.

(1) UDG Buffer pH 8.4: 20 mM Tris, 50 mM KC1, 5 mM MgCl₂ at pH 8.4.

(2) UDG Buffer pH 7.4: 50 mM HEPES-KOH Buffer (pH 7.4), 1 mM

dithiothreitol, 1 mM EDTA, 10 mM NaCl.

(3) UDG Buffer pH 7.8 (HEPES): 70 mM HEPES (pH 8.0), 50 mM EDTA, 1 mM

dithiothreitol (pH 7.8).

(4) UDG Buffer pH 7.8 (Tris): 60 mM Tris (pH 8.0), 1 mM EDTA, 1 mM

dithiothreitol (pH 7.8). Oligonucleotides

Oligodeoxyribonucleotides were obtained from Operan Technologies (Alameda,

CA). Chimeric oligonucleotides were obtained from Oligos Etc. Inc. (Wilsonville, OR) or

synthesized in-house at NeXstar Pharmaceuticals Inc. on an ABI 384 Synthesizer (1 μmole

scale) using standard phosphoramidite chemistry on solid support and deprotected prior to

use.

B. Methods

All UDG reactions were monitored by anion exchange chromatography using

Dionex Nucleopac PA- 100 (4 x 250 mm) Part No. 43010 strong anion exchange column

comprised of methacrylate microbead with quaternary amine ion exchange sites

electostatically attached to inert highly crosslinked 12.5 μm resin. Temperature: 80°C,

flow: 1.5 mL/min. The following two gradient systems were used.

Sodium Chloride Gradient

Mobile Phase: (A) 25 mM Trizma, 1 mM EDTA, 10% acetonitrile (pH 7.5); (B) 25 mM

Trizma, 1 mM EDTA, 10% acetonitrile, 1 M NaCl (pH 7.5). Gradient: 0-30 min: 90%

(A)/10% (B) to 10% (A)/90% (B).

Ammonium Chloride Gradient

Mobile Phase: (A) 25 mM Tris, 1% acetonitrile (pH 8.0); (B) 25 mM Tris, 1% acetonitrile,

1M NH₄C1 (pH 8.0). Gradient: 0-1 min: 100% (A)/0% (B); 1-26 min: 100% (A)/0% (B) to

50% (A)/50% (B).

LC-MS Verification

Identification of the products in the UDG digestions were verified by LC-MS using

a Fisons Quattro II (Beverly, MA) Electrospray Mass Spectrometer (MS) in negative ion

mode. A reversed phase column was run in series with the MS. Chromatography conditions: column Cl 8 (2 mm x 250 mm 5μm 100A) run on a Hewlett Packard 1090

Chromatograph at ambient temperature; flow rate: 0.2 mL/min; mobile phase: (A) 100 mM

ammonium acetate (B) acetonitrile; gradient: 0-5 min: 90%(A)/10%(B); 5-50 min:

90%(A)/10%(B) to 50%(A)/50%(B).

Example 1. Digestion of 5K-Methoxy-SPA-PEG-(NH,VdTdUdAdCdGdAdCdGdA with

UDG

(5-amino-C6)dTdUdAdCdGdAdCdGdA (SEQ ID NO:l) was conjugated with

Methoxy SPA 5K Peg at the 5' end as follows: Solution (A) was prepared by combining 33

μL (21.21 μg/μL) (5-amino-C6)dTdUdAdCdGdAdCdGdA and 167 μL of 100 mM sodium

borate (pH 8.5). Solution (B) was prepared by adding 3.675 mg Methoxy SPA 5,000 MW

PEG (Shearwater) to 100 μL dimethylformamide. Solutions (A) and (B) were combined,

vortexed and heated at 90°C for 2 minutes. Chloroform (150 μL) was added, followed by

addition of 50 μL of 3 M sodium acetate. The solution was mixed for 1 minute and then

centrifuged at 14,000 rpm for 10 minutes at room temperature. The supernatant was

removed and 200 μL of 0.5 M sodium acetate was added. The solution was mixed for 1

minute and then centrifuged at 14,000 rpm for 10 minutes at room temperature. The

supernatant was removed and the product, 5K-Methoxy-SPA-PEG-

(NH₂)dTdUdAdCdGdAdCdGdA was dried on a speed vacuum overnight.

The PEGylated material was purified by reverse phase chromatography using a

Waters C18 Deltapak 5 m C18 300A 3.9 x 150mm column (Part No. 325B14B65).

Temperature: 30°C, flow: 1.0 mL/min , wavelength: 260 nm. Mobile Phase: (A) 100 mM

TEAA (pH 7.0). (B) acetonitrile. Gradient: 0-10 min: 95% (A)/5% (B) to 80% (A)/20%

(B); 10-50 min: 80% (A)/20% (B) to 20% (A)/80% (B). UDG digestion was performed on the PEGylated material with a 0.73 unit/μg UDG

to oligonucleotide concentration at 90°C. The concentration of oligonucleotide in solution

was kept constant at 0.1 μg/μL in UDG Buffer. UDG (36.5μL) was combined with 50 μg

(14.5 μL) PEGylated oligonucleotide and 461.14 μL UDG buffer pH 8.4. Aliquots were

removed at the times indicated in Table 1 and analyzed by anion exchange HPLC using the

sodium chloride gradient. The peak retention times observed for the starting material, 5K-

Methoxy-SPA-PEG-(5-amino-C6)dTdUdAdCdGdAdCdGdA and the UDG cleavage

product, 5'-phosphate-dAdCdGdAdCdGdA (SEQ ID NO:2), were 5.1 minutes and 9.7

minutes, respectively.

Example 2. Digestion of Methoxy SPA 5K PEG-(5-amino-C6 dTdUdCdCdAdGdT

dAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAdAdTdGdG3'3'dTdT-3' with UDG

A (5-amino-C6)dT phosphoramidite was added during traditional oligonucleotide

solid phase synthesis to the final base in the sequence to give 5'-(5-amino-C6)dTdUdCd

CdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAdAdTdGdG3'3'dTdT-3'

(NXdU303, NeXstar) (SEQ ID NO:3). NXdU303 was conjugated with Methoxy SPA 5K

PEG via a succinimidyl propionate linker attached to the PEG. The conjugation proceeded

as follows: Solution (A) was prepared by combining 40 μL (5 μg/μL) of 5'-(5-amino-

C6)dTdUdCdCdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAdAdTdGdG3'3'd

TdT-3' and 160 μL 100 mM sodium borate (pH 8.5). Solution (B) was prepared by adding

1.05 mg Methoxy SPA 5,000 MW Peg to 100 μL dimethylformamide. Solutions (A) and

(B) were combined, vortexed and heated at 90°C for 2 minutes. Chloroform (150 μL) was

added, followed by addition of 50 μL of 3 M sodium acetate. The solution was mixed for 1

minute and then centrifuged at 14,000 rpm for 10 minutes at room temperature. The supernatant was removed and 200 μL of 0.5 M sodium acetate was added. The solution was

mixed for 1 minute, then centrifuged at 14,000 rpm for 10 minutes at room temperature.

The supernatant was removed and the product, Methoxy SPA 5K PEG-(5-amino-

C6)dTdUdCdCdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAdAdTdGdG3'3'd

TdT-3' (5KPEG-dU303) was dried on a speed vacuum overnight.

The PEGylated material was purified by reverse phase chromatography using a

Waters C18 Delta Pak 5μ C18 300A 3.9 x 150 mm column. Temperature: 30°C, flow: 1.0

mL/min, wavelength: 260nm. Mobile Phase: (A) 100 mM TEAA (pH 7.0). (B)

Acetonitrile gradient: 0-10 min: 95% (A)/5% (B) to 80% (A)/20% (B); 10-50 min: 80%

(A)/20% (B) to 20% (A)/80% (B).

UDG digestion was done on the PEGylated material with a 0.73 unit/μg UDG to

oligonucleotide concentration at 90°C. The concentration of oligonucleotide in solution

was kept constant at 0.1 μg/μL in UDG Buffer pH 8.4. UDG (36.5 μL) was combined with

50 μg (15 μL) of the PEGylated oligonucleotide 5KPEG-dU303 and 448.5 μL UDG buffer

pH 8.4 and heated in a 90°C heat block. Aliquots were removed at the times indicated in

Table 2 and analyzed by anion exchange HPLC using the sodium chloride gradient

(Methods, Section B). The peak retention times observed for the starting material, 5K-SPA-

PEG-(5-amino-C6) dTdUdCdCdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdG

dTdAdAdTdGdG3'3'dTdT-3' and the UDG cleavage product 5'-phosphate-

dCdCdAdGdTdAdCdAdAdGdGdTdGdCdTdAdAdAdCdGdTdAdAdTd

GdG3'3'dTdT-3' (SEQ ID NO:4) were 16.15 minutes and 19.63 minutes, respectively

(Figure 1). Example 3. Digestion of 5'-dCdAdCdAdGdGfCfUdAdCdGdGdCdAdCdGfUdAdGdAd

GfCdAfUfCdAdCdCdAfUdGdAfUfCfCdTdGdTdG3'3'dT-3' with UDG

The reactivity of UDG on 2'-fluoro-U and 2'-fluoro-C was tested by digesting 5'-

dCdAdCdAdGdGfCfUdAdCdGdGdCdAdCDGfUdAdGdAdGfCdAfUfCdAdCdCdA

iUdGdAfUfCfCdTdGdTdGS dT-S^* (NX1255, NeXstar) (SEQ ID NO:5) with UDG at both

45°C and 90°C at 0.73 units/μg UDG to oligonucleotide concentration. The concentration

of oligonucleotide in solution was kept constant at 0.1 μg/μL in UDG buffer (pH 8.4).

UDG (36.5 μL) was combined with 50 μg (15 μL) oligonucleotide (NX1255) and 448.5 μL

UDG buffer (pH 8.4) and heated in the appropriate heat block and aliquots were removed at

the following times: 45°C (0, 1, 2, 3, 7 and 19 hours) and 90°C (0, 1, 2, 3, 5 and 17 hours).

Identical digestions at 45°C and 90°C were performed where the UDG was replaced by

water. The aliquots were analyzed by anion exchange HPLC using the sodium chloride

gradient (Methods, Section B) and the retention time for the starting material (NEX 1255)

was 23.7 minutes. No degradation was observed in any of the 45°C digestion time points.

Degradation was observed in both samples with and without UDG incubated at 90°C.

Example 4. Digestion of rUdUrArCrGrArCrGrA with UDG

An all RNA oligonucleotide model with a single dU inserted,

rUdUrArCrGrArCrGrA (SEQ ID NO:6) (Oligo Etc. Inc.) was digested with UDG at 45°C

with 3.7 units/μg UDG to oligonucleotide concentration and at 90°C with 0.72 units/μg

UDG to oligonucleotide concentration. The concentration of oligonucleotide in solution

was kept constant at 0.1 μg/μL in UDG buffer (pH 8.4).

Conditions for the experiment performed at 45°C: 37 μL UDG was combined with

19.7 μg (29 μL) oligonucleotide and 48.29 μL UDG buffer (pH 8.4). Aliquots were removed at the times indicated in Table 3 and analyzed by anion exchange HPLC using the

sodium chloride gradient (Methods, Section B). The retention times for the starting

material, (rUdUrArCrGrArCrGrA) and the UDG cleavage product 5'-phosphate-

rArCrGrArCrGrA (SEQ ID NO:7) were 11.5 minutes and 10.9 minutes, respectively.

Conditions for experiment performed at 90°C: 14.4 μL UDG was combined with 10

μg (14.71 μL) oligonucleotide and 156.6 μL UDG buffer pH 8.4. Aliquots were removed at

1, 2, 3, 4, 5 and 21 hours and analyzed by anion exchange HPLC using the sodium chloride

gradient (Methods, Section B). The retention time demonstrated for the starting material

(rUdUrArCrGrArCrGrA) was 11.4 minutes. Only progressive non-enzymatic degradation

was observed.

Example 5. Digestion of dTdUdTrArCrGrArCrGrA

An RNA oligonucleotide model with an additional dTdUdT on the 5' end,

dTdUdTrArCrGrArCrGrA (Oligo Etc. Inc.) (SEQ ID NO:8) was digested in UDG buffer pH

7.8 (Tris). The oligonucleotide concentration was kept constant at 0.02 μg/μL. Conditions:

UDG was combined with 5 μg oligonucleotide and 242 μL UDG buffer pH 7.8 (Tris) in the

amounts indicated in Table 4. The reactions were placed in a heating block at 37°C for a

specified time and then were placed in a heating block at 65°C. Aliquots were removed

after the reactions had been at 65°C at the times indicated in Table 4 and analyzed by anion

exchange HPLC using the ammonium chloride gradient (Methods, Section B). The

retention times for the starting material, dTdUdTrArCrGrArCrGrA and the UDG cleavage

product 5'-phosphate-dTrArCrGrArCrGrA (SEQ ID NO: 9) were 19.1 minutes and 18.2

minutes, respectively (Figure 2). Example 6. Digestion of dUdTrArCrGrArCrGrA with UDG

An RNA oligonucleotide model with dUdT on the 5' end, dUdTrArCrGrArCrGrA

(SEQ ID NO: 10) (NeXstar) was digested in UDG buffer pH 7.8 (Tris). The oligonucleotide

concentration was kept constant at 0.02 μg/μL. Conditions: 25 μL UDG was combined

with 5 μg oligonucleotide and 250 μL of UDG buffer pH 7.8 (Tris). The reaction was kept

in a heating block at 37°C for 3 hours and then placed in a heating block at 65°C. Aliquots

were removed at the times indicated in Table 5 and analyzed by anion exchange HPLC

using the ammonium chloride gradient (Methods, Section B). The retention times for the

starting material, dUdTrArCrGrArCrGrA and the UDG cleavage product 5'-phosphate-

dTrArCrGrArCrGrA (SEQ ID NO: 11 ) were 18.2 minutes and 18.5 minutes, respectively

Figure 3). In addition, the apyrimidal intermediate, ribose-dTrArCrGrArCrGrA was

observed at 18.0 minutes.

Example 7. Digestion of 5'-r5-amino-C6-dUdTrGrGrArGfUfCfUfUrArGrGfCrAr

GfCrGfCrGfUfUfUfUfCrGrArGfCfUrAfCfUfCfC3'3'dT

A (5-amino-C6) phosphoramidite was added via traditional oligonucleotide solid

phase chemistry at the 5' terminal location in a sequence to give 5'-(5-amino-C6-

dUdTrGrGrArGfUfCfUfUrArGrGfCrArGfCrGfCrGfUfUfUfUfCrGrArGfCfUrAfCfUfCfC

3'3'dT (NX21918, NeXstar) (SEQ ID NO:12). NX21918 was digested in UDG buffer pH

7.8 (Tris). The oligonucleotide concentration was kept constant at 0.02 mg/mL.

Conditions: 50 mL of UDG (1 unit/mL) was combined with 10 mg of NX21918 and 447.98

mL of UDG buffer pH 7.8 (Tris). The reaction was kept at 37°C for 3 hours and then placed

in a heating block at 65°C for 20 hours. The digestion was analyzed by anion exchange HPLC using the ammonium chloride gradient (See Methods, Section B) and the results

indicated that all of the starting material had been converted to pdUdTrGrGrArGfUfCfUfU

rArGrGfCrArGfCrGfCrGfUfUfUfUfCrGrArGfCfUrAfCfUfCfC3'3'dT (SEQ ID NO: 13).

Mass spectrometry analysis verified these chromatographic results.

Example 8. Digestion of 40K PEG-(5-amino-C6 TdUdTfCmGmGrArAfUfCmAmGf

UmGnιAmAfUmGfCfUfUrnAfUnιAfCmAfUfCfCmG3'3'dT

A (5-amino-C6) dT phosphoramidite was added via traditional oligonucleotide solid

phase synthesis to the final base in a sequence to give (5-amino-C6)dTdUdTfCmGmGrAr

AfUfCmAmGfUmGmAmAfUmGfCfUfUmAfUmAfCmAfUfCfCmGS'S'dT (SEQ ID

NO: 14). The amine at the 5' end of this oligonucleotide was reacted with 40K PEG via a

succinimidyl propionate linker. The resulting 40KPEG-(5-amino-C6)dTdUdTfCmGmGr

ArAfUfCmAmGfUmGmAmAfUmGfCfU fUmAfUmAfCmAfUfCfCmG3'3'dT (NX22022,

NeXstar) was digested with UDG buffer pH 8.4 at 90°C and at 45°C. The oligonucleotide

concentration was kept constant at 0.1 mg/mL. Conditions: 365 mL of UDG (1 unit/mL)

was combined with 500 mg of NX22022 and 4,317 mL of UDG buffer pH 8.4. The

reaction was kept at 90°C for 6 hours. Aliquots were removed at 1, 2, 3, 4, 5 and 6 hours

and analyzed by anion exchange HPLC using the ammonium chloride gradient (Methods,

Section B). The chromatographic results indicated that in addition to cleavage of NX22022

at the dU, that NX22022 was also cleaved at the rA positions. Greater than 85% of the full

length material was converted to the UDG cleavage and rA cleavage products. Conditions

at 45°C: 74 mL of UDG (1 unit/mL) was combined with 20 mg of NX22022 and 113.28 mL

of UDG buffer pH 8.4. The reaction was kept at 45°C for 48 hours. The digestion was

analyzed by anion exchange HPLC using the ammonium chloride gradient (Methods, Section B). The chromatographic results indicated that greater than 85% of NX22022 was

converted to the UDG cleavage product. No cleavage at the rA positions was observed.

Table 1. UDG Digestion (Example 1)

Table 2. UDG Digestion of 5KPEG-dU 303 (Example 2)

Table 3. UDG Digestion of SEQ ID NO:6 (Example 4)

Table 4. UDG Digestion of SEQ ID NO:8 (Example 5)

Table 5. UDG Digestion of SEQ ID NO: 10 (Example 6)

Claims

1. A covalent conjugate comprising the formula A-B-C, wherein A is a nucleic

acid component; B is a deoxyuridine; and C is a separable unit, wherein the nucleic acid

component and the separable unit are covalently bonded to the deoxyuridine, provided that

when A is ribonucleic acid, B further comprises a deoxyribonucleotide residue incorporated

between the dU and the ribonucleic acid component.

2. The covalent conjugate of claim 1 wherein the nucleic acid component is a

nucleic acid ligand.

3. The covalent conjugate of claim 2 wherein said nucleic acid ligand is

identified as a ligand of a given target from a candidate mixture of nucleic acids according

to the method comprising:

a) contacting the candidate mixture with the target, wherein the nucleic acids

having an increased affinity to the target relative to the candidate mixture may be

partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the

candidate mixture; and

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched

mixture of nucleic acids, whereby an increased affinity nucleic acid ligand of a given target

may be identified.

4. The covalent conjugate of claim 3 wherein said method further comprises: d) repeating steps a), b) and c).

5. The covalent conjugate of claim 1 wherein said separable unit is a non-

immunogenic, high molecular weight compound.

6. The covalent conjugate of claim 1 wherein the separable unit is covalently

bonded to the dU via a linker.

7. The covalent conjugate of claim 1 further comprising a therapeutic or

diagnostic agent.

8. The covalent conjugate of claim 5 wherein said non-immunogenic, high

molecular weight compound is selected from the group consisting of polyethylene glycol,

dextran and albumin.

9. The covalent conjugate of claim 5 wherein said non-immunogenic, high

molecular weight compound is polyethylene glycol.

10. The covalent conjugate of claim 1 wherein said separable unit is a lipophilic

compound.

11. The covalent conjugate of claim 10 wherein said lipophilic compound is

selected from the group consisting of cholesterol, dialkyl glycerol, and diacyl glycerol.

12. The covalent conjugate of claim 10 wherein said lipophilic compound is

cholesterol.

13. The covalent conjugate of claim 1 wherein said separable unit is a nucleic

acid.

14. The covalent conjugate of claim 1 wherein said deoxyuridine is located at the

5' or the 3' terminus of said nucleic acid.

15. The covalent conjugate of claim 1 wherein the pharmacokinetic properties of

said covalent conjugate are improved relative to the nucleic acid alone.

16. The covalent conjugate of claim 3 wherein the target of said nucleic acid

ligand is an intercellular target.

17. The covalent conjugate of claim 3 wherein the target of said nucleic acid

ligand is an intracellular target.

18. The covalent conjugate of claim 17 wherein the cellular uptake of said

nucleic acid ligand is enhanced relative to the nucleic acid ligand alone.

19. A method of analyzing a nucleic acid component of a covalent conjugate

comprising the formula A-B-C, wherein A is a nucleic acid component, B is a deoxyuridine,

and C is a separable unit, wherein the nucleic acid component and the separable unit are covalently bonded to the deoxyuridine, provided that when A is ribonucleic acid, B further

comprises a deoxyribonucleotide residue incorporated between the dU and the ribonucleic

acid component, said method comprising treating said covalent conjugate with uracil DNA

glycosylase.

20. The method of claim 19 wherein said nucleic acid component is a nucleic

acid ligand.

21. The method of claim 20 wherein said nucleic acid ligand is identified as a

ligand of a given target from a candidate mixture of nucleic acids according to the method

comprising:

a) contacting the candidate mixture with the target, wherein the nucleic

acids having an increased affinity to the target relative to the candidate mixture may be

partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the

candidate mixture; and

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched

may be identified.

22. The method of claim 21 wherein said method further comprises:

d) repeating steps a), b) and c).

23. The method of claim 19 wherein said separable unit is a non-immunogenic,

high molecular weight compound.

24. The method of claim 23 wherein said non-immunogenic, high molecular

weight compound is selected from the group consisting of polyethylene glycol, dextran and

albumin.

25. The method of claim 23 wherein said non-immunogenic, high molecular

weight compound is polyethylene glycol.

26. The method of claim 19 wherein said separable unit is a lipophilic

compound.

27. The method of claim 26 wherein said lipophilic compound is selected from

the group consisting of cholesterol, dialkyl glycerol, and diacyl glycerol.

28. The method of claim 26 wherein said lipophilic compound is cholesterol.

29. The method of claim 19 wherein said separable unit is a nucleic acid.

30. The method of claim 19 wherein said deoxyuridine is located at the 5' or the

3' terminus of said nucleic acid ligand.

31. A method for the preparation of a covalent conjugate comprising a nucleic

acid ligand covalently bonded to a separable unit via a dU, said method comprising:

a) identifying a nucleic acid ligand from a candidate mixture of nucleic

acids, said nucleic acid ligand being a ligand of a given target identified by the method

comprising: i) contacting the candidate mixture with the target, wherein

nucleic acids having an increased affinity to the target relative to the candidate mixture may

be partitioned from the remainder of the candidate mixture;

ii) partitioning the increased affinity nucleic acids from the

remainder of the candidate mixture; and

iii) amplifying said increased nucleic acids to yield a mixture

enriched in nucleic acids having increased affinity for said given target, whereby a nucleic

acid ligand for said given target may be identified;

b) covalently attaching a dU to the 3 ' or 5' end of said nucleic acid

ligand; and

c) covalently attaching a lipophilic compound or a non-immunogenic,

high molecular weight compound to said dU.

32. The method of claim 31 wherein said separable unit is a non-immunogenic,

high molecular weight compound.

33. The method of claim 32 wherein said non-immunogenic, high molecular

albumin.

34. The method of claim 32 wherein said non-immunogenic, high molecular

weight compound is polyethylene glycol.

35. The method of claim 31 wherein said covalent conjugate further comprises a

therapeutic or diagnostic agent.

36. The method of claim 31 wherein said separable unit is a lipophilic

compound.

37. The method of claim 31 wherein said separable unit is covalently bonded to

said dU via a linker.

38. The method of claim 36 wherein said lipophilic compound is selected from

the group consisting of cholesterol, dialkyl glycerol and diacyl glycerol.

39. The method of claim 36 wherein said lipophilic compound is cholesterol.

40. The method of claim 31 wherein said separable unit is a nucleic acid.

41. The method of claim 31 wherein the pharmacokinetic properties of said

covalent conjugate are improved relative to the nucleic acid ligand alone.

42. A method of treating a disease comprising: a) identifying a nucleic acid ligand to a target associated with a

disease and preparing a covalent conjugate, said covalent conjugate comprising a nucleic

acid ligand covalently bonded to a separable unit via a dU, provided that when said nucleic

acid ligand is ribonucleic acid, said covalent conjugate further comprises a

deoxyribonucleotide residue incorporated between the dU and the nucleic acid ligand,

wherein said target is an intercellular target;

b) introducing a pharmaceutical composition of the covalent

conjugate into a patient; and

c) administering uracil DNA glycosylase.

43. The method of claim 42 wherein said nucleic acid ligand is identified as a

comprising:

a) contacting the candidate mixture with the target, wherein the nucleic

partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the

candidate mixture; and

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched

may be identified.

44. The method of claim 43 wherein said method further comprises:

d) repeating steps a), b) and c).

45. The method of claim 42 wherein said separable unit is a non-immunogenic,

high molecular weight compound.

46. The method of claim 42 wherein the separable unit is covalently bonded to

the dU via a linker.

47. The method of claim 42 further comprising a therapeutic or diagnostic agent.

48. The method of claim 45 wherein said non-immunogenic, high molecular

albumin.

49. The method of claim 45 wherein said non-immunogenic, high molecular

weight compound is polyethylene glycol.

50. The method of claim 42 wherein said separable unit is a lipophilic

compound.

51. The method of claim 50 wherein said lipophilic compound is selected from

the group consisting of cholesterol, dialkyl glycerol, and diacyl glycerol.

52. The method of claim 50 wherein said lipophilic compound is cholesterol.

53. The method of claim 42 wherein said separable unit is a nucleic acid.

54. The method of claim 42 wherein said deoxyuridine is located at the 5' or the

3' terminus of said nucleic acid ligand.

55. A method of treating a disease comprising;

a) identifying a nucleic acid ligand to a target associated with a

acid ligand is ribonucleic acid, said covalent conjugate further comprises a

wherein said target is an intracellular target and wherein said separable unit is a carrier

capable of transporting said covalent conjugate across a cell membrane; and

b) introducing said covalent conjugate into a patient.

56. The method of claim 55 wherein said nucleic acid ligand is identified as a

comprising:

a) contacting the candidate mixture with the target, wherein the nucleic

partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the

candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched

may be identified.

57. The method of claim 56 wherein said method further comprises:

d) repeating steps a), b) and c).

58. The method of claim 55 wherein said separable unit is a non-immunogenic,

high molecular weight compound.

59. The method of claim 55 wherein the separable unit is covalently bonded to

the dU via a linker.

60. The method of claim 55 further comprising a therapeutic or diagnostic agent.

61. The method of claim 58 wherein said non-immunogenic, high molecular

albumin.

62. The method of claim 58 wherein said non-immunogenic, high molecular

weight compound is polyethylene glycol.

63. The method of claim 55 wherein said separable unit is a lipophilic

compound.

64. The method of claim 63 wherein said lipophilic compound is selected from

the group consisting of cholesterol, dialkyl glycerol, and diacyl glycerol.

65. The method of claim 63 wherein said lipophilic compound is cholesterol.

66. The method of claim 55 wherein said separable unit is a nucleic acid.

67. The method of claim 55 wherein said deoxyuridine is located at the 5' or the

3' terminus of said nucleic acid ligand.

68. A method for detecting the presence or absence of a target molecule in a

sample which may contain said target molecule comprising:

a) exposing said sample which may contain said target molecule to a

covalent conjugate, said covalent conjugate comprising a nucleic acid ligand covalently

bonded to a separable unit via a dU, provided that when said nucleic acid ligand is

ribonucleic acid, said covalent conjugate further comprises a deoxyribonucleotide residue

incorporated between the dU and the nucleic acid ligand, wherein a complex between said

target molecule and said nucleic acid ligand is formed; and

b) determining whether said complex is formed, whereby the presence

or absence of the target molecule in the sample can be detected.

69. The method of claim 68 wherein said separable unit comprises a biotin, an

enzyme, or a fluorophore.

70. The method of claim 68 wherein the separable unit is covalently bonded to

the dU via a linker.

71. The method of claim 68 further comprising a therapeutic or diagnostic agent.

72. The method of claim 68 wherein said separable unit is a nucleic acid.

73. The method of claim 68 wherein said deoxyuridine is located at the 5' or the

3' terminus of said nucleic acid ligand.

74. A method of claim 68 wherein said nucleic acid ligand is identified as a

comprising: a) contacting the candidate mixture with said target molecule, wherein

nucleic acids having an increased affinity to said target relative to the candidate mixture

may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of

the candidate mixture; and

c) amplifying the increased affinity nucleic acids to yield a ligand-

enriched mixture of nucleic acids, whereby an increased affinity nucleic acid ligand may be

identified.

75. The method of claim 79 further comprising: d) repeating steps a), b) and c).