WO1995030775A1 - High-affinity ligands of insulin receptor antibodies, tachykinin substance p, hiv integrase and hiv-1 reverse transcriptase - Google Patents

Abstract

Methods are described for the identification and preparation of high-affinity ligands to insulin receptor antibodies, substance P, HIV integrase and HIV-Reverse Transcriptase. Included in the invention are specific RNA ligands to insulin receptor antibodies, substance P and HIV intregrase identified by the SELEX method. Also included in the invention are ssDNA ligands to HIV-1 reverse transcriptase identified by the SELEX method. Also included are RNA ligands that inhibit the binding of MA-20 to the human insulin receptor, RNA ligands that are inhibitors of HIV integrase and ssDNA ligands that are inhibitors of HIV-1 reverse transcriptase.

Description

HIGH-AFFINITY LIGANDS OF INSULIN RECEPTOR ANTIBODIES, TACHYKININ SUBSTANCE P, HIV INTEGRASE AND HIV-1 REVERSE TRANSCRIPTASE

This work was partially supported by grants from the United States Government funded through the National Institutes of Health. The U.S. Government may have certain rights in this invention. This work was also supported in part by the Jane Coffin Childs Memorial Fund.

FIELD OF THE INVENTION

Described herein are methods for identifying and preparing high-affinity nucleic acid ligands to insulin receptor antibodies, tachykinin substance P (SP) , Human Immunodeficiency Virus (HIV) integrase, and HIV-1 reverse transcriptase (RT) . For the purposes of this application, HIV integrase includes HIV Type 1 (HIV-1) integrase and HIV integrases that are substantially homologous thereto. By substantially homologous it is meant a degree of amino acid sequence homology of 80% or greater. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Specifically, nucleic acid ligands are described to insulin receptor antibodies, SP, HIV-1 integrase, and HIV-1 RT. Specifically disclosed herein are high-affinity RNA ligands to human insulin receptor antibodies that bind to an epitope within amino acids 450-601 of the alpha subunit of the human insulin receptor. The invention includes high-affinity RNA ligands which inhibit insulin receptor antibody binding. Also disclosed herein are high-affinity RNA ligands to SP and HIV-1 integrase. The invention includes high-affinity RNA inhibitors of HIV integrase. The invention further includes high-affinity single- stranded DNA ligands to HIV-1 RT. Also disclosed are high-affinity ssDNA inhibitors of HIV-1 RT and suicide inhibitors of HIV-1 RT. BACKGROUND OF THE INVENTION

The vertebrate immune system protects organisms from disease by first recognizing and then responding to pathogens, thereby eliminating them. Immune responses are primarily produced by leucocytes (white blood cells), of which there are two main types: (1) phagocytes, which non-specifically bind to micororganisms, internalize them and destroy them and (2) lymphocytes, T cells and B cells, which specifically recognize pathogens.

B cells respond to pathogens by producing protein antibodies, also called immunoglobulins (lg) , which circulate in the blood stream and specifically bind to the foreign antigen which induced them. The antigen may be a molecule on the surface of the pathogen or a toxin which the pathogen produces. T cells, on the other hand, do not produce antibodies, but rather respond to antigens via the T cell receptor (TCR) . At least four different reactions are carried out by T cells. Helper T cells (T_H) assist in B cell development and interact with phagocytic cells to assist in the destruction of pathogens, cytotoxic T cells (T_c) recognize cells infected by viruses and destroy them, and suppressor T cells (T_s) suppress the responses of specific cells. T cells produce their effect by production of cytokines, soluble proteins which signal other cells, or by direct cell-cell interaction.

Ideally, the immune system recognizes components of the organism it protects as self and thus, does not produce an immune response against its own body tissues. Autoimmune diseases, however, are the result of a failure of an organism's immune system to avoid recognition of self, due to production of autoantibodies and auto reactive T cells. The attack by the immune system of host cells can result in a large number of disorders including neural diseases such as multiple sclerosis and myasthenia gravis, diseases of the joints, such as rheumatoid arthritis, attacks on nucleic acids, as observed with systemic lupus erythematosus, and such other diseases associated with various organs, as psoriasis, juvenile onset diabetes, Sjόgren's disease, and Graves disease. Autoimmunity has been implicated in several diseases associated with the human insulin receptor. The human insulin receptor is a tetrameric protein consisting of two extracellular c- subunits containing insulin binding sites, and two transmembrane β subunits with tyrosine kinase activity (Schaefer et al . (1992) J. Biol. Chem. 267 -23393) . Numerous monoclonal antibodies have been developed to the extracellular domain of the human insulin receptor (Kull et al .

(1983) J. Biol. Chem. 25_8.:6561-6566; Morgan and Roth (1986) Biochemistry 25_..1364-1371; Soos et al . (1986) Biochem. J 2^5:199-208; Forsayeth et al . (1987) J. Biol. Chem 26_2_.:4134-4140) . Several of these antibodies are potent inhibitors of insulin binding. In addition, autoimmune patients with extreme insulin resistance (type B) produce autoantibodies that inhibit insulin binding to its receptor (Flier et al . (1975) Science 190:63-65; Flier et al . (1976) J. Clin. Invest. 58.:1442-1449; Kahn et al . (1977) J. Clin. Invest.

.60_.:1094-1106; Taylor et al . (1989) Endocrinol. Metab. Clin. North Am. 18_.:123-143) . Type B insulin resistance is a syndrome that is usually associated with autoimmune diseases, such as acanthosis nigricans and hyperinsulinemia (Kobayashi (1992) Nippon-Naibunpi- Gakkai-Zasshi £8(1) :11) .

Zhang and Roth (1991) Proc. Natl. Acad. Sci. USA 8:9858-9862 used chimeric receptors to identify the epitope(s) recognized by the inhibitory monoclonal antibodies and the autoimmune antibodies. The antibodies studied included 12 monoclonal antibodies (83-7', 3D-7, 18-44, αIR-1, MA-5, MA-10, MA-20, 25-49, 83-14, 47-9, 5D9, MC51) as well as 15 patients' sera with autoimmune anti-insulin receptor antibodies. All of the patients' sera and all 8 monoclonal antibodies that inhibit insulin binding (MA-5, MA-10, MA-20, 25- 49, 83-14, 47-9, 5D9, and MC51) were found to recognize an epitope contained within amino acid residues 450-601 of the subunit of the receptor.

Autoimmunity to the insulin receptor may also contribute to the pathophysiology of insulin-dependent diabetes mellitus (IDDM) (Maron et al . (1983) Nature

303 : 817-818) . Maron et al . found anti-insulin receptor antibodies of the IgM class in the sera of several IDDM patients before treatment with exogenous insulin. These autoantibodies appear to be distinct from the autoantibodies associated with acanthosis nigricans (Maron et al . (1983) Nature 3_03_:817-818) .

RNA sequences capable of binding to a rabbit antibody have been isolated from a complex mixture of RNA transcripts containing synthetically randomized segments (Tsai et al . (1992) Proc. Natl. Acad. Sci. USA £9:8864-8868) . The antibody used in this study was generated by immunizing a rabbit with a 13 amino acid peptide. This work differs from the work described herein in several ways. First, the Tsai et al . antibody recognizes a short peptide which is not expected to adopt any tertiary structure. The selection described herein involves an antibody which recognizes a large protein with a complex tertiary structure, the human insulin receptor. Second, the antibody employed by Tsai et al . is from a rabbit and has no medical relevance. In contrast, the antibodies employed in the selection described herein recognize a medically relevant protein, the human insulin receptor, and the human autoimmune antibodies to insulin receptor employed herein are medically relevant themselves.

Finally, the RNA selected by Tsai et al . binds to the rabbit antibody, but they present no evidence that the RNA structurally resembles the antigen peptide structure. In contrast the selected RNAs described herein cross react with human autoantibodies which recognize the human insulin receptor. This result suggests that the selected RNAs structurally mimic the complex protein epitope on the insulin receptor. This is unexpected and not predicted by the work of Tsai et al . Applicants are unaware of nucleic acid ligands that have been generated that 1) bind specifically to antibodies that target the insulin receptor, 2) bind specifically to any autoantibody or other medically relevant antibody, 3) bind specifically to any antibody which recognizes a complex protein structure or 4) structurally mimic any protein structure including those which serve as antigens.

The peptide substance P (SP) is an eleven amino acid peptide (Fig. 8, SEQ ID NO:23) that belongs to the tachykinin family of neuropeptides. Known mammalian tachykinins (neurokinins) include neurokinin A, neurokinin B, neuropeptide K, and neuropeptide g. All tachykinins share the carboxy-terminal sequence Phe- Xaa-Gly-Leu-Met-NH₂ (where Xaa is an aromatic or aliphatic amino acid) (SEQ ID NO:78) . The mammalian tachykinins are produced by neurons in the central and peripheral nervous system where they are predominantly localized in the nerve terminals (Escher, E. and Regoli, D. (1989) in Peptide Hormones as Prohormones: Processing, Biological Activity. Pharmacology (Martinez, J., ed.) pp 26-52, Ellis Horwood Limited, West Sussex, England) .

Neurotransmitter and neuromodulator functions of SP include peripheral vasodilation, smooth muscle contraction, pain transmission (nociception) , stimulation of exocrine secretions, and immunomodulation (for a review see Escher, E. and

Regoli, D. (1989) in Peptide Hormones as Prohormones: Processing, Biological Activity. Pharmacology (Martinez, J., ed.) pp 26-52, Ellis Horwood Limited, West Sussex, England) . There is^' also evidence that SP has memory-modulating and reinforcing effects. Huston et al . (1993) Psychopharmacology 112:147-162 have suggested a possible link between SP and the impairment in associative functioning accompanying Alzheimer's disease.

The pharmacological importance of substance P is further indicated by recent studies that suggest that SP has a role in angiogenesis ( e . g. , Fan, T. et al . (1993) Brit. J. Pharmacol. 110:43-49) . Fan et al . suggest that the positive interaction between SP and the cytokine interleukin-1 alpha (IL-la) may be important in the angiogenic cascade leading to a variety of diseases characterized by excessive neovascularization ( e . g. , rheumatoid arthritis, atherosclerosis, diabetic retinopathy and cancer) . Blocking substance P activity, therefore, may effectively reduce the progression of the disease. The causative agent for Acquired Immunodeficiency

Syndrome (AIDS) is the Human Immunodeficiency Virus (Gallo et al . (1983) Science 220:865-867; Barre- Sinoussi et al . (1983) Science 220 :868-870 ; Shaw et al . (1984) Science 2^6:1165-1171) . Like all known retroviruses, HIV must reverse transcribe its RNA genome and integrate the double-stranded DNA copy into the host genome (for review; Wong-Staal (1990) In Virology 2nd Ed. (B.N. Fields et al . eds) Raven Press, N.Y. pp. 1529-1543; Cann and Chen (1990) In Virology 2nd Ed. (B.N. Fields et al . eds) Raven Press, N.Y. pp. 1501-1527; Vaishnav and Wong-Staal (1991) Ann. Rev. Biochem. £0:577-630) . The viral component that is essential for formation of a provirus is the integrase protein (Schwartzberg et al . (1984) Cell 37:1043-1052; Donehower and Varmus (1984) Proc. Natl. Acad. Sci. USA £1:6461-6465; Craigie et al . (1990) Cell £2:829-837) . Integrase, in vi tro, has been shown to be necessary and sufficient for processing of the double-stranded viral DNA (processing or donor cut; Katzman et al . (1989) J. Virol. £3:5319-5327; Sherman and Fyfe (1990) Proc. Natl. Acad Sci. USA £7:5119-5123) , cleaving recipient DNA and ligating processed DNA to it (joining or strand transfer; Grandgenett et al . (1986) J. Virol. £8:970- 974; Bushman and Craigie (1991) Proc. Natl. Acad. Sci. USA £8:1339-1343) and for an event that is yet to be demonstrated in vivo, resolution of integrated DNA to component parts (disintegration; Chow et al . (1992) Science 255 :723-726 ; Chow and Brown (1994) J. Virol. £8:3896-3907) . The protein has been divided into three structural domains. The N-terminal domain is highly conserved among retroviral integrases and encodes a Zn++ finger-like DNA binding motif. While the C- terminal domain is variable but consistently basic, with a net charge of about +11. Integrase associates with the double-stranded HIV DNA to form a pre- integration complex which is transported into the nucleus of infected cells (Bowerman et al . (1989) Genes and Development 3_:469-478; reviews: Goff (199.2) Ann. Rev. Genet. 2£:527-544; Whitcomb and Hughes (1992) Ann. Rev. Cell Biol. £:275-306) . It has been suggested that integrase encodes a nuclear localization signal in the C-terminal domain. Mutational analysis of the different domains and the results from complementation tests suggest that integrase functions as a multimer rather than a monomer (Jones et al . (1992) J. Biol. Chem. 267:16037-16040; Engelman et al. (1993) EMBO J. 12_:3269-3275; Leavitt et al . (1993) J. Biol. Chem.

2£8:2113-2119) . This may explain how this enzyme is able to cleave different DNA sequences and remain associated with multiple ends of DNA at the same time. A SELEX-like process was used by Bartel et al . to identify the important structural features of the viral RNA element bound by the Rev protein of HIV-1. (Bartel et al . (1991) Cell £7:529-536.) In one of three rounds of selection performed, wild-type RNA was included in the reaction mixture to compete with the pool RNA for binding to the target protein.

The reverse transcriptase (RT) of Type 1 Human Immunodeficiency Virus (HIV-1) plays an indispensable role in the life cycle of the virus. Its premier function is the synthesis of a double-stranded DNA copy of the RNA genome for integration into the host chromosome. This is achieved by the concerted application of a number of innate activities including minus-strand DNA synthesis via an RNA-dependent DNA polymerase activity, concomitant degradation of the template RNA strand via an RNase H activity, and plus- strand DNA synthesis via a DNA-dependent DNA polymerase activity (Baltimore, D. (1970) Nature

226 :1209; Temin, H. M. and Mizutani, S. (1970) Nature 22£:1211; Gilboa, E. et al . (1979) Cell 18:93-100; Goff, S. P. (1990) J. Acq. Imm. Defic. Syndr. 3_:93-100; Peliska, J. A. and Benkovic, S. J. (1992) Science 2£8:1112-1118) . Because the cells HIV-1 infects contain no endogenous RT, it must also possess a mechanism to ensure its packaging into the mature viral particle to guarantee its presence in the succeeding infection. HIV-1 is generally accepted as the etiological agent of Acquired Immune Deficiency Syndrome (AIDS) . The importance of its function in the life cycle of HIV-1 and the lack of a natural function in the host cell make RT a preferred target for antiviral agents. Several types of HIV-1 RT inhibitors are known.

Many, such as AZT (3 ' -azido-2' , 3' -dideoxythymidine) , are nucleoside analogs, which when incorporated into polynucleotides by HIV-1 RT, result in chain termination. (Kedar, P. S. et al . (1990) Biochem, 2£:3603-3611; Huang, P. et al . (1990) J. Biol. Chem. 265:11914-11918) . Other nucleoside analogs that inhibit HIV-1 RT include ddC (2' , 3 ' -dideoxycytidine) and ddl (2' , 3' -dideoxyinosine) . Inhibitors that are not nucleoside analogs have also been described. These include dipyridodiazepinones ( e . g. , Merluzzi, V. J. et al . (1990) Science 250:1411-1413; Kopp, E. B. et al . (1991) Nuc. Acids Res. 19.(11) :3035-3039) , tetrahydro- imidazo [4, 5, 1-jk] [1,4] -benzodiazepin-2 (IH) -one and - thione (TIBO) derivatives (e.g., Pauwels, T. et al. (1990) Nature 343 :470-474) , and catechin derivatives { e . g. , Nakane, H., and Ono, K. (1990) Biochem. 29:2841- 2845) . These nonnucleosides inhibit by mechanisms other than direct competition for substrate binding sites (Kopp, E. B. et al . (1991) Nuc. Acids Res. 19(11) :3035-3039) .

A family of phosphorodithioate-linked ssDNA nucleotides have been described with the property of inhibiting HIV-1 RT activity at K_t values ranging from 0.5 - 180 nM (Marshall and Caruthers, (1993) , Science 2£9:1564-1570) . The specific sequences of these nucleotides were based on the sequence of various nucleic acid substrates of HIV-RT.

RNA pseudoknots that bind specifically to the polymerase active site of HIV-1 RT and inhibit the RNA- dependent DNA polymerase activity have already been identified using SELEX (U.S. Patent Application No. 07/964,624, which is specifically incorporated herein by reference; Tuerk, C. et al . (1992) Proc. Natl. Acad. Sci., U.S.A. £9:6988-6992) .

A method for the in vi tro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by Exponential enrichment, termed 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, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled Nucleic Acid Ligands, now United States Patent No. 5,270,163 (see also PCT/US91/04078) , each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to as the SELEX Patent

Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.

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 theme, 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 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 high affinity nucleic acid ligands to the target molecule.

The basic SELEX method may be modified to achieve 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, 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 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, 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, 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 delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific 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, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2' -amino modifications . 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'-0-methyl (2'-OMe) . Each of these applications is specifically incorporated herein by reference.

The development of high affinity ligands capable of inhibiting insulin receptor antibodies from binding an epitope contained within residues 450-601 of the alpha subunit of the receptor would be useful in the treatment of type B insulin resistance. Herein described are high affinity nucleic acid ligands which inhibit the binding of insulin receptor antibodies to the epitope contained within residues 450-601 of the a subunit of the insulin receptor.

The development of high affinity ligands of SP are useful as diagnostic and pharmacological agents. Specifically, ligands capable of inhibiting SP would be useful in the treatment or monitoring treatment (i.e., diagnostic applications) of numerous diseases, including angiogenic diseases such as rheumatoid arthritis, atherosclerosis, diabetic retinopathy, and cancer. Herein described are high affinity nucleic acid ligands of SP. Considering the size of SP, it can be assumed that ligands of SP will be inhibitors of SP.

The development of high affinity ligands capable of inhibiting HIV integrase would be useful in the treatment of Human Immunodeficiency Virus. Herein described are high affinity RNA ligand inhibitors of HIV integrase.

The development of high affinity DNA ligands capable of inhibiting HIV-1 reverse transcriptase would be useful in the treatment of Type 1 Human Immunodeficiency Virus. Herein described are high affinity ssDNA ligand inhibitors of HIV-1 reverse transcriptase.

BRIEF SUMMARY OF THE INVENTION The present invention includes methods of identifying and producing nucleic acid ligands and the nucleic acid ligands so identified and produced. Nucleic acid sequences are provided that are ligands of insulin receptor antibodies, tachykinin substance P (SP) , HIV integrase, and HIV-1 RT. For the purpose of this application, HIV integrase includes HIV Type 1 (HIV-1) integrase and HIV integrases that are substantially homologous thereto. By substantially homologous it is meant a degree of amino acid sequence homology of 80% or more. Included within the invention are RNA sequences that are capable of binding specifically to antibodies that target the insulin receptor, SP, and HIV integrase. In particular, RNA sequences are provided that are capable of binding specifically to antibodies that target an epitope contained within residues 450-601 of the alpha subunit of the human insulin receptor. Additionally, ssDNA sequences are provided that are capable of binding specifically to HIV-1 RT.

Specifically included within the invention are the RNA ligand sequences shown in Figures 2 (SEQ ID NOS: 4- 15) , 9 (SEQ ID NOS: 26-42) , 10 (SEQ ID NOS: 43-75) , Table 4 (SEQ ID NOS: 84-138) , and the ssDNA ligand sequences shown in Figures 18 (SEQ ID NOS: 150-186) and 19 (SEQ ID NOS: 187-216) .

Also included in this invention are nucleic acid ligands of insulin receptor antibodies that inhibit antibody binding to the insulin receptor. Specifically, RNA ligands are identified and described which inhibit the binding of human insulin receptor antibodies to an epitope contained within residues 450- 601 of the alpha subunit of the human insulin receptor. Also included in this invention are RNA ligands of HIV integrase that are inhibitors of HIV integrase. Specifically, RNA ligands are identified and described which inhibit the viral DNA processing or encapsidation activities of HIV integrase. Also included in this invention are DNA ligands of HIV-1 RT that are inhibitors of HIV-1 RT. Specifically, ssDNA ligands are identified and described which inhibit the RNA- dependent DNA polymerase activity of HIV-1 RT.

Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to insulin receptor antibodies, SP, HIV integrase, and HIV-1 RT, comprising the steps of (a) preparing a candidate mixture of nucleic acids, (b) contacting the candidate mixture of nucleic acid ligands with insulin receptor antibodies, SP, HIV integrase, or HIV-1 RT, wherein nucleic acids having an increased affinity to insulin receptor antibodies, SP, HIV integrase, or HIV-1 RT relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to insulin receptor antibodies, SP, HIV integrase, or HIV-1 RT may be identified.

Also included in the invention is the method of identifying nucleic acid ligands and ligand sequences to HIV integrase described above wherein the mixture contacted includes non-amplifiable random pool nucleic acids.

Also included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to SP in solution comprising the steps of (a) preparing a candidate mixture of nucleic acids; (b) partitioning between members of said candidate mixture on the basis of affinity to immobilized SP; (c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to immobilized SP; (d) determining which of the nucleic acids from step (c) demonstrates binding to SP in solution; (e) selecting the nucleic acid ligand from the ligand mixture (d) that has the highest affinity for binding to SP in solution; (f) preparing a second candidate mixture of nucleic acids by mutagenizing the nucleic acid ligand selected in (e) ; and (g) repeating steps (b) , (c) , and (d) . The mutagenesis allows the evolution of a more favorable primary sequence solution to higher order structures, since the initial experiment contained 10¹⁴ unique sequences, which is only a minute fraction of the 4⁶⁰ possible sequences 60 nucleotides in length.

More specifically, the present invention includes the RNA ligands to insulin receptor antibodies, SP, HIV integrase, and ssDNA ligands to HIV-1 RT identified according to the above-described method(s) , including those ligands listed in Figures 2, 9, 10, 18, 19, and Table 4. Also included are RNA ligands to insulin receptor antibodies and HIV integrase that are substantially homologous to any of the given ligands and that have substantially the same ability to bind and inhibit the antibodies to the insulin receptor and

HIV integrase. Further included in this invention are RNA ligands to insulin receptor antibodies and HIV integrase that have substantially the same structural form as the ligands presented herein and that have ..substantially the same ability to bind insulin receptor antibodies and HIV integrase and inhibit the antibodies **^"'from binding to the insulin receptor and HIV integrase.

Also included are RNA ligands to SP that are substantially homologous to any of the given ligands and that have substantially the same ability to bind SP. Further included in this invention are RNA ligands to SP that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind SP.

Also included are ssDNA ligands to HIV-1 RT that are substantially homologous to any of the given ligands and that have substantially the same ability to bind and inhibit HIV-1 RT. Further included in this invention are ssDNA ligands to HIV-1 RT that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind and inhibit HIV-1 RT.

Further included in this invention are ssDNA ligands incorporating at specific positions nucleotide analogs possessing a reactive group able to covalently crosslink the ligand to HIV-1 RT upon binding. This invention also includes the ligands as described above, wherein covalent crosslinking is coupled to the activity of the HIV-1 RT.

The present invention also includes modified nucleotide sequences based on the RNA and DNA ligands identified herein and mixtures of the same.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the 40N template oligo (SEQ ID NO:l) , the upstream PCR and cloning primer (SEQ ID

NO:2) , and the downstream PCR and cloning primer (SEQ ID NO:3) used in SELEX experiments to select RNA ligands to insulin receptor antibodies.

Figure 2 shows the selected sequences to insulin receptor antibodies isolated from the library after cycle 11 (SEQ ID NOS:4-15) . The highly conserved twenty-one nucleotide sequence is shown within the box (where R=purine and Y=pyrimidine) . The top of the Figure shows the 40N region with the fixed sequences. Only the 40 positions originally randomized are shown below for each numbered individual. The full-length sequence includes the upstream and downstream sequences as shown at the top of the Figure.

Figure 3 is an autoradiogram showing specific binding of RNAs #1 (SEQ ID NO:4) and #9 (SEQ ID NO:12) to MA-20 antibody. Lanes A-E show body-labeled RNA #1 (lane A) immunoprecipitated with MA-20 antibody (lanes B and E) , with normal mouse IgGs (lane C) , or without any antibodies (lane D) . Lanes F-J show body-labeled RNA #9 (lane F) immunoprecipitated with MA-20 antibody (lanes G and J) , with normal mouse IgGs (lane H) , or without any antibodies (lane I) . The immunoprecipitated RNAs were visualized by polyacrylamide gel electrophoresis (PAGE) upon a 10% gel with urea.

Figure 4 shows the percent of RNA bound versus MA- 20 antibody concentration (in nM) at three different RNA concentrations: 10 pM (O) , 20 pM (□) , and 40 pM (Δ) .

Figure 5 shows that RNA #9 (SEQ ID NO:12) blocks the binding of MA-20 to the human insulin receptor. For samples A-E, the ectodomain of the human insulin receptor was preincubated with ¹²⁵I-insulin. Sample F contained only ¹²⁵I-insulin and no insulin receptor. MA-20 or 83-7 anti-insulin receptor antibody was added in the presence or absence of competitor RNAs (samples A-F) . The amount of ¹²⁵I that was precipitated was calculated. The values shown are the average of those obtained from an experiment performed in triplicate. The error bars represent the standard deviation. A sample containing no antibody was used to determine the background level of ¹²⁵I-insulin found in the pellets. The numbers shown have been corrected by this value (180 CPM) .

Figure 6A is an autoradiogram showing that all three of the autoimmune serum samples (B10 in lane D, B7 in lane E, and Bd in lane F) immunoprecipitate the selected RNA sequence (RNA #1; SEQ ID N0:4) . The autoradiogram also shows that neither the nonimmunized mouse IgGs (lane B) nor the normal human serum (lane C) precipitate RNA #1. Figure 6B is an autoradiogram showing that all three of the autoimmune serum samples (BIO in lane D, B7 in lane E, and Bd in lane F) immunoprecipitate the selected RNA sequence (RNA #9; SEQ ID NO:12) . The autoradiogram also shows that neither the nonimmunized mouse IgGs (lane B) nor the normal human serum (lane C) precipitate RNA #9. Figure 6C is an autoradiogram showing the results of a competition assay. Lanes A-C contain BIO patient serum. Lanes D-F contain normal human serum. RNA #9 was added with either no competitor RNA (lanes A and D) , unlabeled RNA #9 (lanes B and E) , or unlabeled pool RNA (lanes C and F) .

Figures 7A and B show the predicted RNA secondary structure of portions of selected RNAs #1 (SEQ ID NO:16) (7A) and #9 (SEQ ID NO:17) (7B) , which contain three stem regions (labeled I, II, and III) . The nucleotides in the shaded region originate from the fixed flanking sequence surrounding the selected variable RNA sequence.

Figure 8 shows the starting ssDNA template and RNAs, PCR primers, and peptides used in SELEX experiments to select RNA ligands to SP. For Cys-SP, "Ac" indicates that the peptide was synthesized with an acetylated N-terminus. (SEQ ID NOS-.18-25) .

Figure 9 shows sequences from the 6ON regions of

RNA ligands selected to SP in SELEX experiment A (SEQ ID NOS:22, 26-42) . The full-length sequence includes the upstream and downstream fixed sequences as shown at the top of the Figure. Sequence numbers are preceded by the letter "A" to designate their selection in experiment A. The number of identical sequences among the 33 clones analyzed is indicated in parenthesis next to the sequence number. Groups 1-4 each represent a single "parental" sequence with variants resulting from point mutations presumably introduced by the poly erases used in the SELEX protocol. All unique sequences were placed in group 5. For sequences listed below the first sequences in groups 1-4, only differences from the first sequence are shown. Nucleotides listed below gaps in the first sequence indicate insertions. The hyphen in the ligand A14 sequence denotes the deletion of this nucleotide.

Figure 10 shows alignments of sequences from the 6ON (selected) regions of experiment B ligands to SP (SEQ ID NOS:43-75) . The full-length sequence includes the upstream and downstream fixed sequences as shown at the top of the Figure. The ligands have been assigned to three classes on the basis of sequence and secondary structure similarities. Secondary structure similarities were determined by phylogenetic comparison (Fox, G., and Woese, C. (1975) Nature

25£:505-507; Noller, H. F., and Woese, C. R. (1981) Science 212 :403-410) . Sequence numbers are preceded by the letter B to designate their selection in experiment B. Nucleotide positions are numbered consecutively from a starting position dictated by aligned consensus sequences. Gaps in the sequences represent the absence of nucleotides at those numbered positions. K_d measurements from single-point equilibrium dialysis binding experiments are shown to the right of the individual ligands. Binding reactions consisted of 20 nM ³H-SP (P₀) and either 0.2 μM RNA (a) or 1.0 μM RNA (b) . ND = not determined. Nucleotide frequencies at the indicated positions are shown below the class 1 and 2 alignments.

Figure 11 shows binding curves for ligand A13 (SEQ ID NO:22) (Δ) , experiment B selection cycle 12 pooled RNAs (■) , and ligand B28 (SEQ ID NO:53) (•) . The fraction of ³H-substance P bound in equilibrium dialysis experiments is plotted as a function of total RNA concentration. In each experiment, the concentration of substance P applied to one side of the dialysis membrane (P₀) was 20 nM. All binding reactions were at room temperature in IX substance P binding buffer.

Figure 12A shows predicted secondary structures for ligands A13 (SEQ ID NO:22) and B28 (SEQ ID NO:53) . The 23-nucleotide 5' and 25-nucleotide 3' fixed sequences complementary to the PCR primers are shown in bold italicized type. Figure 12B shows consensus sequences and predicted secondary structures for the highly conserved regions of experiment B class 1 (SEQ ID NO:76) and 2 (SEQ ID NO:77) ligands. Universally conserved nucleotides are shown as normal capital letters. Lower case letters are used to indicate positions were a specific nucleotide is not universally conserved but occurs at a frequency of > 90%. The following symbols are used to indicate other nucleotide patterns at individual locations: N = any base; R = A or G; Y = C or TJ; W = A or U; V = A , C, or G; K = G or U; D = A, G, or U; H = A, C, or U; ^••• = base-pairing is sometimes, but not always, possible between these two nucleotide positions. In the class 2 consensus structure, the terminal seven nucleotides of the 5' fixed sequence are shown in bold italicized type.

Figure 13 shows competition between substance P fragments and intact substance P for binding to ligand B28 (SEQ ID NO:53) . Equilibrium dialysis binding reactions consisted of 1.6 μM ligand B28 RNA, 1.6 μM substance P (P₀; including 20 nM ³H-substance P) , and 32 μM (20-fold excess) of the competing peptide fragment. The fraction of ³H-substance P bound in the absence of added competitor was 0.63. An 86.1 % decrease (inhibition) in ³H-substance P bound was observed in the presence of 32 μM (P₀) competing unlabeled substance P. The percent decrease (inhibition) in ³H-substance P bound in the presence of individual substance P fragments is expressed relative to intact substance P = 100 % inhibition.

Figure 14 shows a plot of percent inhibition of ³H-substance P bound to ligand B28 (SEQ ID NO:53) in the presence of varying concentrations of unlabeled substance P (SEQ ID NO:23) (•) or a peptide (rSP) which contains the same amino acid sequence as substance P but in the reverse orientation (SEQ ID NO:25) (■) . Each equilibrium dialysis binding reaction consisted of 1.6 μM ligand B28 RNA and 1.6 μM of substance P (P₀; including 20 nM ³H-substance P) in addition to the competing peptide at a concentration of 0, 1.6, 6.4, 25, or 100 μM. The percent decrease (inhibition) in the fraction of ³H-substance P bound in the presence of competing peptide is expressed relative to the fraction of ³H-substance P bound in the absence of added competitor (0.69) .

Figure 15A shows the proposed secondary structure of P5 RNA based on data from chemical probing experiments and computer modeling, and Figure 15B shows the proposed secondary structure of A54 RNA based on data from chemical probing experiments and computer modeling.

Figure 16 shows the experimental design and oligonucleotide sequences used in SELEX experiments to select ssDNA ligands to HIV-1 RT. A degenerate double- stranded DNA library was created using the Polymerase

Chain Reaction to amplify oligo 1, using oligos 2 and 3 as primers. Box 1 shows the 35N template oligo (oligo 1) (SEQ ID NO:145) , Box 2 shows the upstream PCR primer (oligo 2) (SEQ ID NO:146) , Box 3 shows the biotinylated downstream PCR primer (oligo 3) (SEQ ID NO:147) and downstream cloning primer (oligo 3 with biotins removed) , Box 4 shows the upstream cloning primer (oligo 4) (SEQ ID N0.148), and Box 5 shows the DNA sequencing primer (oligo 5) (SEQ ID NO:149) .

Figure 17 shows protein excess binding curves measuring affinity of ssDNA library after various SELEX cycles. K_d values were determined using an algorithm to fit the data points to Equation 2 of Example 20.

Figure 18 shows sequences to HIV-1 RT isolated from the library after cycle 12 (SEQ ID NOS:150-186) . The top of the Figure shows the upstream PCR primer (see Fig. 16) and the complement of the downstream PCR primer and downstream cloning primer (see Fig. 16) . Only the 35 positions originally randomized are shown below for each numbered individual. However, the full- length sequence includes the upstream and downstream sequences as shown at the top of the Figure. Isolates were grouped and aligned by common primary sequence elements. Clones are indicated by number. Approximately 3 of 4 selected ligands contained the sequence CCCCT (boxed) , or a variant of this pentamer. Other regions of similarity among isolates are shaded. Because these ligands were sequenced using a primer that annealed adjacent to the 35N region, often the sequence of the first few nucleotides at the 3' end was indecipherable. As the sequences of the unreadable regions are not necessary for this analysis, they are represented by "N"s.

Figure 19 shows sequences to HIV-1 RT isolated from the library after cycle 15 (SEQ ID NOS:187-216) . The top of the Figure shows the upstream PCR primer (see Fig. 16) and the complement of the downstream PCR primer and downstream cloning primer (see Fig. 16) . Only the 35 positions originally randomized are shown below for each numbered individual. However, the full- length sequence includes the upstream and downstream sequences as shown at the top of the Figure. Isolates were grouped and aligned by common primary sequence elements. Clones are indicated by number. Isolates were grouped and aligned by common elements. CCCCT, or a variant of this pentamer, is shown as boxed. Other regions of similarity among isolates are shaded.

Figures 20A-20H show the predicted secondary structures of eight individual ligands (RT1 (SEQ ID N0.215) , RT4 (SEQ ID NO:208) , RT6 (SEQ ID NO:201) , RT8 (SEQ ID NO:204) , RT10 (SEQ ID NO:200) , RT12 (SEQ ID NO:192) , RT26 (SEQ ID NO:188) , and RT36 (SEQ ID NO:211)) . The structure of each of the eight ligands in this figure include elements common to many other members of its respective group (boxed or shaded as in Fig. 19) . The 35 positions originally randomized are demarcated by vertical lines.

Figures 21A-21E show the conserved internal loop motif. The sequence and predicted secondary structure of the internal loop motif of ligands RT26 (SEQ ID NO:188) and RT1 (SEQ ID NO:215) is illustrated, along with variants of the motif found in ligands RT4 (SEQ ID NO:208) , RT8 (SEQ ID NO:204), and RT36 (SEQ ID NO:211) . The conserved loop sequences are indicated in boldface. The stems closing each side of the internal loop vary in both sequence and length.

Figures 22A-22C show the protein excess binding curves of selected individuals. The percent of ligand bound is plotted as a function of total protein concentration. The dissociation constants of the RNA pseudoknot (RNA pk) and the degenerate library (RO) are shown in Figure 22A. The dissociation constants of RTl

(SEQ ID NO-215) , RT4 (SEQ ID NO:208) , RT6 (SEQ ID NO:201) , RT8 (SEQ ID NO:204) , RT10 (SEQ ID NO:200) , RT12 (SEQ ID NO-192), RT26 (SEQ ID NO:188) , and RT36

(SEQ ID NO:211) are shown in Figures 22B and 22C. Dissociation constants were determined as in Example 20.

Figure 23 shows the proposed secondary structure of the RNA pseudoknot inhibitor (SEQ ID NO:217) (Tuerk, C. et al . (1992) Proc. Natl. Acad. Sci., U.S.A. £9:6988-6992) .

Figure 24 shows the products of intramolecular extension of RT26 (SEQ ID NO:188) . End-labeled RT26 was extended with a saturating concentration of either HIV-1 RT, AMV RT, or Sequenase.

Figures 25A-25I show the inhibition of RNA- dependent DNA polymerase activity of HIV-1 RT. The substrate for the inhibition assay is shown in Figure 25A. Extension reaction products are shown for RO (degenerate ssDNA library) , RNApk (RNA pseudoknot) , RTl (SEQ ID NO:215) , and RT26 (SEQ ID NO:188) in Figures 25B, D, F, and H. The K plots are also shown in Figures 25C, E, G, and I.

Figures 26A and 26B show the sequences of individuals isolated from the biased randomization

SELEX of RTl (SEQ ID NOS:220-235) . The 35N positions, aligned with the "wild-type" sequence of RTl are shown in Figure 26A. Positions absolutely conserved are indicated with an open circle, those partially conserved (fewer than three individuals possess a substitution) with a triangle, and those preferring a substitution with a bullet. Complementary sequences able to form secondary structure interactions are underlined. Predicted secondary structure of RTl with a consensus sequence (suggested by the results of A) replacing the "wild-type" 35N region is shown in Figure 26B (SEQ ID NO:236) . Only the upstream invariant region and 35N region are shown. Variable positions are represented with an N. The two preferred substitutions (G₂ and T₁₈) are indicated in boldface.

Figures 27A-27C show the predicted secondary structures of RTl (SEQ ID NO:215) and truncates ligand RTlt49 (SEQ ID NO:237) and ligands RTlt30 (SEQ ID NO:238) .

Figure 28 shows the inhibition specificity assay.

Inhibition of the RNA-dependent DNA polymerase activity of three reverse transcriptases (HIV-1 RT, AMV RT, and MMLV RT) was performed as described in Example 20, with inhibitor RTlt49 (SEQ ID NO:237) present at the indicated concentrations in nM.

Figure 29 shows the competitive binding of RTl (SEQ ID NO-.215) and the RNA pseudoknot (RNA pk) (SEQ ID NO:217) .

Figure 30 shows the way in which covalent crosslinking is coupled to the activity of the enzyme. Step 1 shows the catalytic addition of a nucleotide triphosphate to a ligand that has a nucleotide analog at its 3' end containing an electron withdrawing group (EWG) at the 2' carbon. Step 2 shows the spontaneous elimination event whereby the newly added nucleotide is released and yields an electrophilic carbon at the 3' position of the sugar that is stabilized by the electron withdrawing group at the 2' position. Step 3 shows the formation of a covalent crosslink between the protein and the ligand. DETAILED DESCRIPTION OF THE INVENTION

This application describes high-affinity nucleic acid ligands to insulin receptor antibodies, SP, HIV integrase and HIV-1 RT identified through the method known as SELEX. The SELEX method is described in U.S. Patent Application Serial No. 07/536,428, entitled Systematic Evolution of Ligands by Exponential Enrichment, now abandoned, U.S. Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled Nucleic Acid Ligands, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled Nucleic Acid Ligands, now United States Patent No. 5,270,163, (see also PCT/US91/04078) . These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.

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 by 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 to 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 partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate an enriched candidate mixture. The SELEX Patent Applications also describe ligand solutions obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.

The methods described herein and the nucleic acid ligands identified by such methods are useful for both therapeutic and diagnostic purposes. Therapeutic uses include the treatment or prevention of diseases or medical conditions in human patients. Therapeutic uses may also include veterinary applications. Diagnostic utilization may include both in vivo or in vi tro diagnostic applications. The SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications. SELEX identifies nucleic acid ligands that are able to bind targets with high affinity and with surprising specificity. These characteristics are, of course, the desired properties one skilled in the art would seek for in a diagnostic ligand. The nucleic acid ligands of the present invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any nucleic acid ligand by procedures known in the art to incorporate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures. The nucleic acid ligands to insulin receptor antibodies, SP, HIV integrase and HIV-1 RT described herein may specifically be used for identification of insulin receptor antibody, SP, HIV integrase and HIV-1 RT proteins. SELEX provides high affinity ligands of a target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to the specific target of insulin receptor antibodies, SP, HIV integrase, and HIV-1 RT. In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligands to insulin receptor antibodies, SP, HIV integrase and HIV- 1 RT are described.

In order to produce nucleic acids 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 have the highest possible affinity to the target.

In co-pending and commonly assigned U.S. Patent Application Serial No. 07/964,624, filed October 21, 1992 ('624) , methods are described for obtaining improved nucleic acid ligands after SELEX has been performed. The '624 application, entitled Methods of Producing Nucleic Acid Ligands, is specifically incorporated herein by reference.

The following definitions are provided to clarify their meaning in the specification and the claims. An autoantibody is an antibody that acts against the cellular components of the organism in which it is formed.

A monoclonal antibody is a single pure antibody produced in quantity by a cultured clone of a special type of cell called a B lymphocyte.

As used herein, antibody refers to either a monoclonal antibody or an autoantibody. In the present invention, a SELEX experiment was performed in search of nucleic acid ligands with specific high affinity for the human insulin receptor monoclonal antibody (MA-20) from a degenerate library containing 40 random positions (40N) (Example 1) .

After 11 rounds of selection, two clones were chosen for further study, and both were found to specifically bind to the MA-20 antibody (Example 2) . One clone was tested and found to have a K_d of 2 nM (Example 2) . Furthermore, one clone was tested and found to block binding of MA-20 to the human insulin receptor by interacting with a site similar or identical to that of receptor binding (Example 2) . Additionally, the two clones selected for further study were recognized by autoimmune sera from patients with severe insulin resistance type B (Example 3) . Labeled RNA and unlabeled RNA from one clone were found to compete in the presence of autoimmune serum (Example 3) . Secondary structure of selected ligands was predicted by computer analysis (Example 4) .

Another SELEX experiment was performed in search of a nucleic acid ligand with specific high affinity for SP from a degenerate library containing 60 random positions (6ON) (Example 6) . A SELEX procedure was used to isolate RNAs that bind substance P immobilized on a solid support (Example 7) . RNAs that also bind substance P in solution were identified and the tightest binder was subjected to mutagenesis in a second SELEX procedure to evolve ligands with a higher affinity for the peptide (Example 8) . A comparative analysis of 36 ligands isolated from the second SELEX experiment revealed two main sequence classes with highly conserved secondary structures within each class (Example 9) . Dissociation constants for the interaction of these ligands with SP in solution were determined by equilibrium dialysis. The amino acid residues involved in the interaction with the highest affinity ligand (190 nM K_d) were mapped by determining which of a_. set of overlapping fragments of substance P can compete with the intact peptide for binding (Example 10) . A binding competition experiment also demonstrated the ability of the same ligand to discriminate between substance P and the reverse orientation of the same amino acid sequence (Example 11) . The results from this study demonstrate that SELEX can yield high affinity RNA ligands to small non-constrained peptides.

In the present invention, a SELEX procedure was also used to isolate RNAs with specific high affinity for HIV-1 integrase from a degenerate library containing 30 random positions (3ON) (Examples 13 and 14) . Secondary structure of selected ligands was predicted by computer analysis and chemical and enzymatic structure analysis (Example 15) . RNA truncate studies of a selected ligand were performed to determine the minimal binding domain of the RNA (Example 16) . In vi tro inhibition of integrase is demonstrated in Example 17. A binding competition experiment demonstrated the ability of a selected ligand (P5) to be a potent competitive inhibitor of HIV-1 integrase (Example 18) . In the present invention, a SELEX experiment was also performed in search of single-stranded DNA ligands with specific high affinity for HIV-1 RT from a degenerate library containing 35 random positions (35N) . A large family was identified with an apparent affinity for HIV-1 RT about 700 times higher than the library from which they originated (described in Examples 20 and 21) . At least seven members of this diverse family, sharing little similarity with each other or with the RNA pseudoknot at the levels of primary and secondary structure, inhibit the RNA- dependent DNA polymerase activity of HIV-1 RT at very low concentrations, possibly competing with substrate for the polymerase active site by virtue of their higher affinity for RT (described in Example 21) . For at least one inhibitor this inhibition is specific for HIV-1 RT, as the polymerase activity of reverse transcriptases from Avian Myeloblastoma Virus (AMV-RT) and Moloney Murine Leukemia Virus (MMLV-RT) were unaffected by the presence of the inhibitory DNA ligand RTlt49 (SEQ ID NO:237) . For one of the ssDNA inhibitors (RTl) (SEQ ID NO:215) , the importance of each selected residue was assessed by introducing an average of 9 new mutations (in the originally randomized region) and selecting for variants maintaining high affinity (described in Example 23) . Based on these results, we then removed 40% of the ligand and observed only a moderate loss of affinity. The 5' half of the truncate contained an internal loop motif common to other members of the selected library, likely creating a helix bend that provides a specific shape for direct contact by HIV-1 RT. The truncated ligand inhibited the polymerase activity of HIV-1 RT as well as the full-length ligand (see Example 23) , and binding of the truncate and the RNA pseudoknot were mutually exclusive (see Example 24) , suggesting they interact with HIV-1 RT at a common site. A phosphorothioate cap added to the 3' end of truncate RTlt49 results in stability against 3' to 5' exonucleases without affecting binding affinity of the ligand for HIV-1 RT.

This invention includes the specific nucleic acid ligands shown in Figures 2, 9, 10, 18, 19, and Table 4. These figures and table include RNA ligands to human insulin receptor antibodies (Figure 2; SEQ ID NOS:4- 15) , RNA ligands to SP (Figures 9 and 10; SEQ ID NOS:22, 26-75) , RNA ligands to HIV-1 integrase (Table 4; SEQ ID NOS:84-138), and DNA ligands to HIV-1 RT

(Figure 18, SEQ ID NOS: 150-186; and Figure 19, SEQ ID NOS:187-216) identified by the SELEX method described herein. This invention further includes RNA ligands which inhibit the binding of human insulin receptor antibodies to an epitope contained within residues 450- 601 of the alpha subunit of the human insulin receptor. This invention further includes ssDNA ligands of HIV-1 RT that are inhibitors of HIV-1 RT. The scope of the ligands covered by this invention extends to all nucleic acid ligands of insulin receptor antibodies, SP, HIV integrase, and DNA ligands of HIV-1 RT, modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to the nucleic acid ligand sequences shown in Figures 2, 9, 10, 18, 19, and Table 4. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%.

A review of the sequence homologies of the RNA ligands shown, for example, in Figures 9 and 10 for SP, Table 4 for HIV-1 integrase, and Figures 18 and 19 for HIV-1 RT shows that sequences with little or no primary sequence homology may have substantially the same ability to bind SP, HIV-1 integrase, and HIV-1 RT, respectively. For these reasons, this invention also includes nucleic acid ligands that have substantially the same structure and ability to bind insulin receptor antibodies, SP, HIV integrase, HIV-1 RT as the nucleic acid ligands shown in Figures 2, 9, 10, 18, 19 and Table 4. Substantially the same ability to bind insulin receptor antibodies, SP, HIV integrase, and

HIV-1 RT means that the affinity is within one to two orders of magnitude of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence -- substantially homologous to those specifically described herein -- has substantially the same ability to bind insulin receptor antibodies, SP, HIV integrase, or HIV- 1 RT .

This invention also includes the ligands as described above, wherein certain chemical modifications are made in order to increase the in vivo stability of the ligand or to enhance or mediate the delivery of the ligand. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions of a given nucleic acid sequence. See, e.g., Cook et al . PCT Application WO 92/03568; U.S. Patent No. 5,118,672 of Schinazi et al . ; Hobbs et al . (1973) Biochem. 12_.:5138; Guschlbauer et al . (1977) Nucleic Acids Res. 4_.:1933; Shibahara et al . (1987) Nucleic Acids Res. .15:4403; Pieken et al . (1991) Science 253 :314 , each of which is specifically incorporated herein by reference. For example, modifications at the 2' position of the sugar ( e . g. , replacement of a H at the 2' position with a chloro, fluoro, or O-methyl) may provide resistance to intracellular or extracellular endon cleases. Additionally, a 3' cap consisting of three or four nucleotides that are connected with phosphodithioate bonds could provide resistance for DNA ligands against 3' - 5' exonucleases. Such modifications may be made post-SELEX (modification of previously identified unmodified ligands) or by incorporation into the SELEX process. Example 25 describes post-SELEX modification of a ssDNA ligand to contain a 3' cap consisting of four thymine residues linked by a phosphorothionate backbone. The phosphorothionate cap added stability without affecting binding affinity of the ligand for its target.

Additionally, the ligands can be modified by mutagenesis either during the SELEX process or post- SELEX to yield ligands with better properties. PCR mutagenesis is described in detail below, however, any mutagenesis process known to one of ordinary skill in the art can be applied in a similar manner. The RNA ligands to the insulin receptor antibodies described herein are useful as pharmaceuticals. This invention, therefore, also includes a method for the treatment of autoimmune diseases, such as extreme insulin resistance type B, and IDDM by administration of a nucleic acid ligand capable of binding to the autoimmune anti-insulin receptor antibodies.

The DNA ligands to the HIV-1 RT protein described herein are useful as pharmaceuticals and as part of gene therapy treatments. According to methods known to those skilled in the art, the nucleic acid ligands may be introduced intracellularly into cells infected with the HIV virus, where the nucleic acid ligand will compete with the substrate for the nucleic acid binding site and/or polymerase active site. As such, transcription of HIV genes can be prevented.

The invention also includes the ligands as described above, wherein nucleotide analogs are incorporated at a specific position, and further that these nucleotide analogs possess a reactive group which is able to covalently crosslink the ligand to .HIV-1 RT upon binding. This invention also includes the ligands as described above, wherein covalent crosslinking is coupled to the activity of the HIV-1 RT. As described above, because of their ability to selectively bind HIV integrase, the nucleic acid ligands to HIV integrase described herein are useful as pharmaceuticals. This invention, therefore, also includes a method for the treatment of HIV by administration of a nucleic acid ligand capable of binding to the HIV integrase.

Therapeutic compositions of the nucleic acid ligands may be administered parenterally by injection, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, 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 parenteral 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 the formulations containing RNA ligands for systemic delivery may be via subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectal suppository.

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 experimental procedures used to generate high-affinity nucleic acid ligands to antibody MA-20. Example 2 describes the RNA ligands to MA-20. Example 3 describes the recognition of the RNA ligands by human autoimmune sera. Example 4 describes the structural features of the ligands . Example 5 describes the procedures used to generate 2'-NH₂ pyrimidine modified RNA ligands to insulin receptor antibodies. Example 6 describes the experimental procedures used to generate high-affinity nucleic acid ligands to SP. Example 7 describes the RNA ligands to SP. Example 8 describes selecting for higher affinity ligands to SP by mutagenesis . Example 9 describes the predicted secondary structure of selected ligands. Example 10 describes the amino acids required for interaction with ligand B28. Example 11 describes the specificity of ligand B28 for SP over rSP. Example 12 describes the procedures used to generate 2' -NH₂ pyrimidine modified RNA ligands to SP.

Example 13 describes the experimental procedures used to generate high-affinity nucleic acid ligands to HIV-1 integrase. Example 14 describes the RNA ligands to HIV-1 integrase. Example 15 describes the predicted secondary structure of selected HIV-1 integrase ligands. Example 16 describes the truncates of P5 RNA to determine the minimal binding domain of the RNA. Example 17 describes the inhibition of integrase by P5. Example 18 describes the competitive binding of P5 RNA. Example 19 describes the procedures used to generate 2'-NH₂ pyrimidine modified RNA ligands to HIV-1 integrase.

Example 20 describes the experimental procedures used to generate high-affinity ssDNA ligands to HIV-1 RT. Example 21 describes the high-affinity DNA ligands to HIV-1 RT shown in Figures 18 and 19. Example 22 describes suicide inhibitors of HIV-1 RT. Example 23 describes the essential elements of RTl. Example 24 describes the competition between RTl and RNA Pseudoknot for RT binding. Example 25 describes the synthesis of ssDNA ligand with a phosphorothioate cap and reports on its K_d. EXAMPLE 1. EXPERIMENTAL PROCEDURES

This Example provides the general procedures followed and incorporated into Examples 2-5.

Materials. The antibody MA-20 was purchased from Amersham. The human insulin receptor was a generous gift from Drs. Leland Ellis and Erik Shaefer, Institute of Biosciences and Technology, Texas A and M University, Houston, Texas. Monoclonal antibody 83-7 was a generous gift of Dr. Kenneth Siddle, Department of Clinical Biochemistry, University of Cambridge, UK. Serum obtained from three patients diagnosed with extreme insulin resistance (type B) was a generous gift from Drs. Simeon Taylor and Domenico Accili, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland. All other materials were purchased from commercial sources.

SELEX. Essential features of the SELEX protocol have been described in detail in U.S. Patent No. 5,270,163 as well as in previous papers (See, e.g., Schneider et al . (1992) J. Mol. Biol. 228:862) .

Briefly, DNA templates for in vi tro transcription (that contain a region of forty random positions flanked by constant sequence regions) and the corresponding PCR primers were prepared chemically using established solid phase oligonucleotide synthesis protocols.

A library of 10¹⁴ RNA molecules was generated that contained a 40-nucleotide long region of random sequence flanked by defined sequences (Fig. 1; SEQ ID NO:l) . The random region was generated by utilizing an equimolar mixture of the four unmodified nucleotides during oligonucleotide synthesis. The random pool was generated by in vi tro transcription of a synthetic DNA template. For the initial round of selection, 30 μg of RNA was incubated with 1 pmol naive mouse IgGs in a binding buffer containing 30 mM tris-HCl pH 7.5, 10 mM MgCl₂, 2 mM dithiothreitol, 1% bovine serum albumin (BSA) , and 5 units RNasin (Promega) in a total volume of 100 μl . After 30 minutes of incubation at 25°C with gentle shaking, 20 μl of goat anti-mouse IgG coated magnetic beads were added and allowed to incubate an additional 30 minutes at 25°C. The beads were then pelleted and discarded. This step served to remove RNA sequences which bound to the constant region of the antibodies, or which adhered nonspecifically to the magnetic beads used in the immunoprecipitation. The supernatant, which contained the bulk of the RNA pool, was removed to a new tube and incubated with 1 μl of 1 mg/ml mouse monoclonal MA-20 antibody for 30 minutes at 25°C. 20 μl of goat anti-mouse IgG coated magnetic beads were then added and allowed to incubate an additional 30 minutes at 25°C. The beads were then pelleted, and the pellets were washed twice with 0.5 ml of the binding buffer. RNA was eluted from the pellets in 100 μl of 0.1 M EDTA. The eluate was then applied to a Sephadex G-25 spin column to remove EDTA and salts. A 28-nucleotide DNA primer oligonucleotide complementary to the 3' sequence of the original pool RNA (with the addition of several nucleotides, including 6 guanines at the 5' end) (Fig. 1; SEQ ID NO:3) was added to the eluted RNA at a concentration of 1 μM, and reverse transcription was performed using

Avian Myoblastoma Virus (AMV) reverse transcriptase and a Tris buffer from Boehringer Mannheim. After a 30 minute incubation at 37°C, a second DNA primer was added, which is the DNA equivalent of the 5' end of the original RNA pool and contains the 17 nucleotides of promoter sequence for T7 RNA polymerase as well as some additional nucleotides (Fig. 1; SEQ ID NO:2) . 30 cycles of PCR were carried out using Taq DNA polymerase from Perkin-Elmer. Amplified DNA was phenol extracted, precipitated in ethanol and resuspended in 20 μl of 10 mM tris-HCl pH 8 and 1 mM EDTA. Half of this solution was used as the template for an in vi tro transcription reaction with T7 RNA polymerase in 100 μl total volume. Subsequent cycles of selection were performed in the same way, except that the pre-binding to naive mouse IgGs was performed only on alternate cycles.

EXAMPLE 2. RNA LIGANDS TO MA-20

After eleven rounds of in vi tro selection, the immunoprecipitated RNAs were reverse transcribed, and the resulting cDNAs were PCR amplified and subcloned (as described supra) . The PCR-amplified cDNAs were cloned into the plasmid vector PUC19 by techniques known in the art, and sequencing of the clones was performed according to the manufacturer's instructions for the Sequenase kit, U.S. Biochemical. Twenty-two clones were sequenced and were found to encode eleven different RNA insert sequences (Fig. 2; SEQ ID NOS:4- 15) . These RNAs all contain a highly conserved twenty- one nucleotide sequence (R=purine and y=pyrimidine) . Several of the RNA sequences were found to be present multiple times. Many of the sequences were shorter than the original 78-nucleotide pool RNAs due to deletions within the randomized region that arose during reverse transcription and PCR amplification.

Binding is specific for the MA-20 antibody. Two of the cloned sequences, #1 (SEQ ID NO:4) and #9 (SEQ ID NO:12) in Figure 2, were chosen for further characterization. The plasmids containing the cDNAs were linearized with restriction enzyme BamHI and transcribed using T7 RNA polymerase and a buffer containing 30 mM tris-HCl pH 8.1, 25 mM magnesium chloride, 10 mM dithiothreitol, .01% Triton X-100, 1 mM spermidine and 2.5 mM each nucleoside triphosphate. α- ³²p _ATp _{was nc}ι_uded in the reaction (2 microliters of 3000 mCi/mmol) . The resulting RNA sequences are referred to herein as body-labeled. The resulting transcripts contain both the selected insert sequences as well as the fixed flanking RNA sequences described supra . The body-labeled RNAs #1 (lanes A-E of Fig. 3) or #9 (lanes F-J of Fig. 3) (1 nM) were incubated in 100 μl^' of binding buffer with the MA-20 antibody (10 μg/ml) (lanes B,E,G, and J of Fig. 3) , with normal mouse IgGs (20 μg/ml) (lanes C and H of Fig. 3) , or without any antibodies (lanes D and I of Fig. 3) for 15 minutes at 25°C with shaking. The antibody-RNA complexes were immunoprecipitated with goat anti-mouse IgG magnetic beads (Dynal) (lanes B-D and G-I of Fig. 3) or with protein G sepharose (Pharmacia) (lanes E and J of Fig. 3) by incubating at 25°C for 15 minutes with shaking and then pulling the beads out of solution with a magnet (lanes B-D and G-I of Fig. 3) or pelleting with beads in microfuge (lanes E and J of Fig. 3) . The pellets were washed two times with 500 μl of binding buffer, and the immunoprecipitated RNAs were eluted with the addition of 10 μl of 500 mM EDTA. Immunoprecipitated RNAs were visualized by polyacrylamide gel electrophoresis (PAGE) upon a 10% gel with urea. As a control, one-half of the amount of the labeled RNAs #1 (lane A) and #9 (lane F) that was added to the immunoprecipitations was electrophoresed. The RNA from both clones #1 and #9 was found to bind to the MA-20 antibody independent of the technique used to immunoprecipitate the antibody complexes (magnetic beads or Protein G-Sepharose beads) . Neither RNA bound to non-immunized mouse IgG antibodies or to the magnetic beads in the absence of antibody. Thus, binding is specific for the MA-20 antibody, suggesting that the RNA binds to the variable region of the antibody.

In a separate experiment, carried out as described supra, clones #3 (SEQ ID NO:6) and #4 (SEQ ID NO:7) were shown to bind MA-20. Calculating K., of a selected RNA ligand. In order to determine an approximate K_d for binding to the MA-20 antibody, ³²P-body-labeled RNA #9 (10, 20, and 40 pM) (Fig. 2; SEQ ID NO:12) was incubated with increasing amounts of MA-20 antibody for 30 minutes at 25°C with shaking. The RNA-antibody complexes were immunoprecipitated with protein G sepharose beads as described above. The pelleted beads were washed with 500 μl of binding buffer and repelleted to determine the amount of radioactivity precipitated using a scintillation counter. Plots of the percent of RNA bound versus antibody concentration at three different RNA concentrations are shown in Figure 4. As a maximum of 40-60% of the total counts were immunoprecipitated even when incubated with extremely high antibody concentrations, plotted numbers have been normalized by these amounts. K_d was obtained from a non-linear least squares fit (Kaleidagraph, Synergy Software, Reading, PA) to the dependence of the observed percent RNA bound to the concentration of antibody (% RNA bound = (maximum bound) (antibody concentration) / (K_d + antibody concentration) ) . The apparent K_d was determined to be approximately 2 nM, based on three independent experiments (as described supra) . This is likely to be an upper estimate of the K_d, as the immunoprecipitation procedure involves several steps including extensive washing to remove nonspecifically bound material.

Hence, the RNA appears to bind extremely tightly to the protein, consistent with the conditions under which the selection was performed.

RNA #9 blocks the binding of MA-20 to the human insulin receptor. Studies of the MA-20 interaction with insulin receptor have revealed that this particular antibody binds to a site near the insulin binding site on the receptor (Forsayeth et al . (1987) J. Biol. Chem. 262 :4134) . Antibody binding is not competitive with insulin binding to the receptor, and in fact, the antibody interaction tends to stabilize the insulin-insulin receptor complex by preventing the dissociation of prebound insulin (Forsayeth et al . (1987) J. Biol. Chem. 262:4134) . We took advantage of this feature of the antibody to determine whether the selected RNA bound at or near the site of receptor binding to the antibody.

Purified ectodomain of the insulin receptor protein was pre-bound to ¹²⁵I-labeled insulin, followed by addition of the MA-20 antibody in the presence or absence of selected RNA #9 (SEQ ID NO:12) , or original pool RNA (Fig. 5) . For samples A-E in Figure 5, the ectodomain of the α subunit of the human insulin receptor (10 nM) was preincubated with ¹²⁵I-insulin (1 nM) in 100 μl of binding buffer at 4°C for 20 minutes with shaking. Sample F of Figure 5 contained only ¹²⁵I- insulin (1 nM) and no insulin receptor. Either the MA- 20 (20 nM) or the 83-7 (1 μl of ascites fluid) anti- insulin receptor antibody was added in the presence or absence of competitor RNAs (RNA #9 at 1 μM or 100 nM; RNA pool at 1 μM) along with protein G sepharose beads and incubated at 4°C for 30 minutes. Next, the beads were pelleted in a microfuge, washed twice with 500 μl of binding buffer, and repelleted to determine the amount of radioactivity in the pellets. The values shown in Figure 5 are the average of those obtained from an experiment performed in triplicate.

In the presence of a 10-fold excess of RNA #9 over receptor, we observed a nearly 90% decrease in the amount of ¹²⁵I-insulin precipitated (sample C of Fig. 5) . With a 100-fold excess of this RNA, the amount of insulin precipitated was not detectable above background counts (sample B of Fig. 5) . In contrast, there was no significant effect observed in the presence of 100-fold excess of the nonselected pool RNA (sample D) . In addition, no inhibitory effect of the #9 RNA was observed in the presence of a different monoclonal antibody, 83-7, which recognizes a different epitope on the insulin receptor protein (Zhang and Roth (1991) Proc. Natl. Acad. Sci. USA £8=9858) (sample E of Fig. 5) . Thus, RNA #9 blocks the binding of MA-20 to the human insulin receptor by interacting with the MA- 20 antibody at a site similar or identical to that of the insulin receptor binding site on the antibody.

EXAMPLE 3. SELECTED RNAS ARE RECOGNIZED BY HUMAN AUTOIMMUNE SERA FROM PATIENTS WITH SEVERE INSULIN RESISTANCE TYPE B In order to explore the possibility that the selected RNA might be a structural mimic of the antigenic epitope of insulin receptor that is recognized by the MA-20 mouse monoclonal antibody, we obtained samples of serum from three patients diagnosed with extreme insulin resistance (type B) . This autoimmune disorder is often characterized by the presence of autoantibodies against the same amino acid region of human insulin receptor that is recognized by the MA-20 mouse antibody (Zhang and Roth (1991) Proc. Natl. Acad. Sci. USA £8=9858) .

Selected RNA #1 (SEQ ID NO:4) (Fig. 6A) or #9 (SEQ ID NO:12) (Fig. 6B) were immunoprecipitated with MA-20 (lane A in Figs. 6A and 6B) , normal mouse IgGs (lane B in Figs. 6A and 6B) , normal human serum (lane C in Figs. 6A and 6B) , or three autoimmune patient sera

(lane D serum BIO, lane E serum B7, and lane F serum Bd) . 1 μl of the MA-20 antibody (2.5 μM), 1 μl of normal mouse IgGs (5 μM) , 30 μl of the normal human serum, or 30 μl of the autoimmune sera were prebound to 20 μl of the protein G sepharose beads in 250 μl binding buffer for 30 minutes at 25°C with shaking. As the serum antibodies are of indeterminate concentration and also contain an abundance of contaminating nucleases, the sera were prebound to protein G- Sepharose beads and washed with binding buffer. The beads were pelleted in a microfuge, washed with 500 μl binding buffer, and repelleted. They were then resuspended in 100 μl of binding buffer containing either selected RNA #1 (100 pM) (Fig. 6A) or #9 (100 pM) (Fig. 6B) end-labeled with ³²P using T4 polynucleotide kinase and [γ-³²P] adenosine triphosphate. The binding reactions were incubated and assayed as described in Example 2. Precipitated RNA was analyzed on denaturing polyacrylamide gels. Figs. 6A and 6B show autoradiograms of these gels. All three of the autoimmune serum samples precipitated the selected RNA sequences. In contrast, neither selected RNA was precipitated by nonimmunized mouse IgGs or by normal human serum.

Competition Assay. In order to determine the specificity of the interaction of antibodies in the human serum samples, a competition assay was performed. Protein G sepharose beads were prebound to either antibodies from B10 patient serum (Fig. 6C, Lanes A-C) or to normal human serum (D-F) as described above. ³ P end-labeled #9 RNA (100 pM) was added with either no competitor RNA (A and D) , unlabeled #9 RNA (1 μM) (B and E) , or unlabeled pool RNA (1 μM) (C and F) , and the binding and analysis of bound RNAs was performed as described above. The B10 autoimmune antibody binding of selected RNA #9 can be specifically but not non- specifically, competed by RNA (Fig. 6C) . Thus, complexes are specifically competed by unlabeled RNA #9, but not by unlabeled pool RNA.

Although sequences for the human autoantibodies are not known, it seems unlikely that they would be identical for all three human patients as well as a mouse monoclonal antibody. Yet all of these antibodies bind to a common antigenic epitope of the human insulin receptor, and all bind to the selected RNA sequence reported herein. These results suggest that the selected RNA sequence structurally mimics a major antigenic epitope of the human insulin receptor. EXAMPLE 4. CHARACTERIZATION OF THE STRUCTURAL

FEATURES OF THE SELECTED RNA

The sequences shown in Figure 2 were theoretically folded using the RNA folding program MulFold, and the most stable predicted secondary structures for portions of RNA #1 (SEQ ID NO:4) and #9 (SEQ ID NO:12) are shown in Figure 7A and B (SEQ ID NOS:16 and 17) . The predicted structures consist of three stems separated by unpaired nucleotides and capped at one end by an 8- nucleotide loop consisting of some of the most highly^' conserved nucleotides in the sequence.

In order to test this predicted secondary structure, several variants were synthesized by transcription off of synthetic DNA templates. The insert sequence from RNA #9 (SEQ ID NO:12) binds MA-20 without the fixed flanking sequences (data not shown) .

EXAMPLE 5. MODIFIED 2 ' -NH- PYRIMIDINE RNA LIGANDS

TO INSULIN RECEPTOR ANTIBODIES In order to generate ligands with improved stability in vivo, an experiment is carried out with randomized RNA containing amino (NH₂) functionalities at the 2'-position of each pyrimidine. A library of 10¹⁴ RNA molecules is generated that contains a 40- nucleotide long region of random sequence flanked by defined sequences. Defined nucleotide sequences in the flanking regions of the template serve as primer annealing sites for PCR. The random nucleotides of the initial candidate mixture are comprised of 2'-NH₂ pyrimidine bases. The rounds of selection and amplification are carried out as described supra in Example 1 using art-known techniques.

To avoid the selection and amplification of undesired RNA molecules that bind to the constant region of the antibodies or which adhere nonspecifically to the magnetic beads used in immunoprecipitation, the newly transcribed pool is incubated with goat antimouse IgG coated magnetic beads before the next round of selection. The procedure is reiterated until the enriched pool of RNA shows significantly improved affinity to the insulin receptor antibody over the initial random pool . The resulting RNA ligands are reverse transcribed and the cDNAs are PCR amplified, subcloned, and sequenced as described supra.

EXAMPLE 6. EXPERIMENTAL PROCEDURES

This Example provides general procedures followed and incorporated into Examples 7-12.

Materials . Synthetic single-stranded DNAs (ssDNA) were obtained from Operon (Alameda, California) . Cys- SP (SEQ ID NO:24) and Cys-rSP (SEQ ID NO:25) were synthesized and purified by Macromolecular Resources (Fort Collins, Colorado) . Thiopropyl-activated Sepharose 6B, SP, and all SP fragments with the exception of SP 1-6 were purchased from Sigma. SP 1-6 was purchased from Peninsula Laboratories, Inc.

(Belmont, California) . [2-L-Prolyl-3,4-³H(N) ] -SP and all radionucleotides were obtained from NEN Research Products (Dupont; Wilmington, Delaware) . Enzymes were purchased from commercial sources. Immobilizing SP on thiopropyl-activated Sepharose.

Cys-SP (Fig. 8, SEQ ID NO:24) was covalently coupled (disulfide bond) to thiopropyl-activated Sepharose 6B through an interaction of the Cys-thiol group of peptides with hydroxypropyl-2-pyridyl disulfide ligands of the matrix. The coupling reaction consisted of 1.4 mg Cys-SP and 1 g pre-swollen thiopropyl-Sepharose in a final volume of 7 ml in the following buffer: 500 mM NaCl, 1 M EDTA, 10 mM Tris-HCl, pH 8.4. The suspension was gently mixed for 1 hr at 4° C, then transferred to a chromatography column and washed with 30 ml of 100 mM sodium acetate, pH 6.0. Remaining hydroxypropyl-2-pyridyl disulfide ligands were reacted by suspending the matrix in 7 ml of 100 mM sodium acetate plus 5 mM 3-mercaptoethanol, followed by gentle mixing at room temperature for 45 min. The matrix was washed in SP binding buffer (10 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM KCl, 5 mM CaCl₂) , and the concentration of bound peptides was quantitated by ninhydrin assays. Storage was in SP binding buffer with 0.1 % sodium azide at 4°C. Thiopropyl Sepharose 6B utilized for counterselection and the dilution of SP-Sepharose was prepared as described above except for the omission of SP.

Random sequence RNA pool. Template DNA for the initial random sequence RNA population was generated from a synthetic random sequence ssDNA pool (Fig. 8, SEQ ID NO:21) . The random region was generated by utilizing a mixture of the four unmodified nucleotides (the molar ratios of which are adjusted to yield a 1:1:1:1 ratio of incorporated nucleotides) during oligonucleotide synthesis. The ssDNAs contained 60 nucleotides of contiguous random sequence flanked by defined 5' and 3' ends that permit primer hybridization (Fig. 8) . Double-stranded DNA (dsDNA) molecules, synthesized initially by klenow enzyme, and subsequently (following cycles of selection) by Taq DNA polymerase, have a T7 RNA polymerase promoter at the 5' end. In vi tro transcription of 500 pmoles (~3 X 10" unique sequences) of dsDNA template yielded the initial pool of uniformly [o.-³²P] GTP-labeled 107-nt random sequence RNAs. SELEX Experiment A. This experiment identifies

RNA ligands which bind to SP. Uniformly ³²P-labeled RNAs were suspended in 25 μl of SP binding buffer, heated at 70°C for 5 min, then cooled to room temperature. The quantity of RNA used for each selection cycle is indicated in Table 1. The RNA suspension was applied to a 100-μl SP-Sepharose column (80 μM SP) at room temperature, followed by 10 200-μl SP binding buffer wash volumes. Peptide-bound RNAs were then recovered with five 200-μl volumes of binding buffer containing 100 mM dithiothreitol (DTT) . DTT reduces the linker disulfide bond resulting in the release of peptide from the matrix. The DTT eluate was extracted once with phenol and the RNAs were recovered by ethanol precipitation with 20 μg of yeast tRNA as carrier. Reverse transcription, PCR amplification, and T7 RNA polymerase transcription were performed essentially as described in (Tuerk, C. and Gold, L. (1990) Science 249:505-510) . Transcription of PCR products yielded the RNA pool for the next cycle of selection and amplification.

Prior to some of the selection cycles, a counterselection procedure was done as indicated in Table 1. The counterselection process entailed applying the RNA suspension to a thiopropyl-Sepharose column (not coupled with SP) and unbound RNA was then applied to the SP-Sepharose column as described above. SELEX Experiment B. This experiment was performed in an attempt to find ligands with a higher affinity to SP by mutagenizing the highest affinity ligand identified in Experiment A. Random mutagenesis (by the PCR mutagenesis procedure described below) of the highest affinity ligand from Experiment A (ligand A13 (SEQ ID NO:22) described in Example 7 below) provided the template DNA used to initiate Experiment B. Uniformly ³²P-labeled RNAs (200 pmoles for each selection cycle) were suspended in 400 μl of 188 mM NaCl, denatured by heating at 90°C for 90 sec, then quick-cooled on ice. After the addition of 100 μl 5X SP binding buffer (minus NaCl) , the RNA was combined with a 100-μl column volume of SP-Sepharose suspended in 400 μl SP binding buffer. SP concentrations (as a function of column volume) used for each selection cycle are listed in Table 2. The 1 ml suspension was mixed on a rocking plateform at room temperature for 30 min. An identical binding procedure was followed for cycles in which a counterselection column (no peptide) preceded the SP-Sepharose column in the selection scheme (indicated in Table 2) . Bound RNAs were pelleted with the counterselection matrix by centrifugation (-1000 x g, 5 sec) and a 100-μl column volume of SP-Sepharose was suspended in the supernatant. Following the 30 min incubation, the SP- Sepharose was pelleted as above, and the supernatant was removed. The matrix was resuspended in 400 μl binding buffer and transferred to a syringe column (shortened 1-ml syringe with a small quantity of glass wool at the bottom) . The flow-through volume was collected and the column was washed with an additional 10 200-μl volumes of binding buffer. Peptide-bound RNAs were eluted with five 200-μl volumes of binding buffer containing 100 mM DTT. RNAs were recovered as in Experiment A and amplified. PCR amplification following selection cycles 1-5 was by the PCR mutagenesis procedure described below. Standard PCR amplification followed selection cycles 6-12.

PCR mutagenesis . Essential features of a modified PCR procedure for the introduction of random point mutations into DNA are well-known in the art and are described in (Cadwell, R. C, and Joyce, G. F. (1992) PCR Methods Appl. 2 :28-33; Bartel, D. P., and Szostak, J. W. (1993) Science 2£1:1411-1418; and Leung, D. W. et al . (1989) Technique .1:11-15) . In Experiment B of this study, the reaction mixture for selection cycle one consisted of 5 pmoles ligand A13 (SEQ ID NO:22) dsDNA

(for selection cycles 2-6, all of the cDNA recovered from the prior selection cycle served as template) , 1 mM dCTP, 1 mM dTTP, 0.2 mM dGTP, 0.2 mM dATP, 7 mM

MgCl₂, 0.55 mM MnCl₂, 100 pmoles of each primer, and 3 units of Tag polymerase (Promega) in 100 μl Tag buffer (supplied with enzyme) . The mixture was subjected to three PCR cycles: each cycle was 93°C, 45 sec; 50°C, 45 sec; and 72°C, 2 min. A 13 μl volume of the reaction was transferred to a new tube and brought up to 100 μl with fresh reaction buffer (same composition minus template) followed by an additional three cycles of PCR mutagenesis. This dilution-PCR mutagenesis procedure was repeated nine more times for a total of 11 3-cycle reactions (33 doublings) . Products from the 11th 3-cycle reaction were amplified further by diluting 13 μl of the reaction in 87 μl of a standard PCR reaction mixture (0.4 mM dNTPs, 3.75 mM MgCl₂, 250 pmoles of each primer, and 5 units of Tag polymerase in IX Tag buffer) followed by 8 PCR cycles . Products from the final reaction were used as template for T7 RNA polymerase transcription. To analyze the mutation results, PCR products from the final amplification of

PCR-mutagenized ligand A13 (for selection cycle 1) were cloned and sequenced.

DNA sequencing. PCR products were cloned into the Hind III and Bam HI restriction sites of pUC18 and sequenced by the dideoxynucleotide termination method using modified T7 DNA polymerase (Sequenase 2.0; United States Biochemical) and universal forward and reverse primers.

Equilibrium dialysis. Equilibrium dialysis experiments were performed with a Spectra/Por"

(Spectrum; Houston, Texas) equilibrium dialyzer, Spectra/Por" (Spectrum; Houston, Texas) microcell dialysis chambers (200-μl chamber volumes) , and 12000- 14000 MWCO dialysis membranes (Spectrum; Houston, Texas) . These membranes allow passage of SP but not 107-nt RNAs. Sample volumes were 200 μl on each side of the membrane (i.e., 200 μl per chamber) . All experiments were at room temperature in IX SP binding buffer. RNAs (unlabeled) were denatured prior to re- equilibration in binding buffer as described above for Experiment B. For binding curves (Fig. 11) , RNA concentrations between 50 nM and 20 μM were utilized. Single point K_d estimates for individual Experiment B ligands were performed with 200 nM or 1 μM RNA. The concentration of ³H-SP applied to one side of the membrane (P_D; this symbol is used throughout this application to denote the peptide concentration as applied to one side of the membrane) was 20 nM for binding curves and single-point K_ά estimates. For binding competition experiments (Figs. 13 and 14) , equal concentrations of the competing peptide were applied to each side of the membrane at the start of dialysis. The dialysis cells were rotated at 10 rpm to shorten the time required to reach equilibrium. Equilibrium dialysis initiated with ³H-SP on one side of the membrane and in the absence of RNA was performed to determine the length of time required to attain equilibrium (~ 2.5 hours for Spectra/Por"-2 membranes) ; dialysis times for binding measurements were at least one hour longer than this determined time. Following equilibration, samples were withdrawn from the dialysis chambers, added to scintillation fluid, and counted. Determining Equilibrium Dissociation Constants. Equilibrium dissociation constants (K_d) were defined by the following equation (Rosen, D. et al . (1980) Biochemistry 11:5687-5692) : K_ά= [RJ [P_£] / [P where R_f is the concentration of unbound RNA (i.e., total RNA minus [P_b] ) , P_£ is the concentration of unbound peptide (concentration on the side of the membrane that does not contain RNA) , and [P_b] is the concentration of peptide bound to RNA ( [P] on side of membrane that contains RNA minus [P_£] ) . Donnan effects were neglected in K_ά measurements because it was assumed that this problem would be overcome by the high NaCl concentration (Karush, F. and Sonnenberg, M. (1949) J. Amer. Chem. Society 21:1369) . The fraction of SP bound was calculated by dividing [PJ by [P , where [P_t] is the total peptide concentration in the dialysis chamber containing RNA.

EXAMPLE 7. RNA LIGANDS TO SP

Ligands generated by Experiment A. RNA ligands with affinity for SP were isolated in SELEX Experiment A described in Example 6 by selecting for RNAs present in a random sequence pool that bind SP immobilized on a solid support . The initial random sequence RNA population for experiment A consisted of approximately 3 X 10" unique molecules, each with 60 nt of contiguous random sequence (SEQ ID NO:21) . RNA 5' and 3' defined ends and their complimentary primer sequences are shown in Figure 8. The dissociation constant of the unselected random sequence RNA pool was roughly estimated at 1.2 mM as indicated by an equilibrium dialysis experiment (see Example 6, supra) with 20 μM RNA and 20 nM (P₀) of ³H-SP where the fraction of SP bound was 0.017. The constraints of the dialysis system prohibited the use of significantly higher concentrations of RNA in the analysis. For the first selection cycle, 500 pmoles of RNA was used; this quantity was reduced as the copy number of individual species increased (200 pmoles in cycle 2 and 50 pmoles for the remaining cycles) . RNA pools subjected to selection cycles 5-12 were first counterselected on thiopropyl-Sepharose 6B (see Example 6) to remove RNAs with affinity for Sepharose or the linker arm. A significant increase in binding to SP was observed after the seventh selection cycle, with only a moderate improvement in subsequent cycles (Table 1) . AMV reverse transcriptase sequencing of RNA pools showed a significant decrease in sequence randomness following selection cycles 7-12 (data not shown) . No additional change in the sequence pattern was observed for RNA pools generated from selection cycles 10-12, suggesting that no further enrichment was occurring under these conditions. PCR products from the 12th cycle of selection and amplification were cloned and sequenced as described in Example 6. Of the 33 clones sequenced, 18 are unique (Fig. 9) (SEQ ID NOS:22, 26-42) . However, 10 of the unique sequences may have resulted from point mutations within a selected sequence during the amplification or cloning procedures (see sequence groups 1-4, Fig. 9) . Representatives of each sequence group were analyzed by equilibrium dialysis for their ability to bind SP in solution. Only group 3 and group 4 ligands (SEQ ID

NOS:22, 35-38) demonstrated binding to free SP at RNA and peptide concentrations of 4 μM and 2 μM (P_D) , respectively. Ligand A13 (SEQ ID NO:22) , with an estimated K_ά of 14 μM under these conditions, exhibited the highest affinity for SP in solution. The K_d for ligand A13 was subsequently more accurately estimated at 5.8 μM with a five-point binding curve (Fig. 11) , an improvement in affinity of about 200-fold over the initial unselected random sequence RNA pool. Sequence groups which did not exhibit binding to free SP under these conditions were presumably selected for their affinity to an SP conformation that is more prevalent when the peptide is coupled to the matrix, or to an epitope that includes portions of both the peptide and the linker arm. Alternatively, ligands with affinity for the linker arm alone may have escaped the counterselection process resulting in their subsequent elution with DTT.

EXAMPLE 8. SELECTING FOR HIGHER AFFINITY LIGANDS TO

SP BY MUTAGENESIS

Ligands Generated by Experiment B. Higher affinity ligands to SP were produced and isolated in Experiment B described in Example 6. Using ligand A13 dsDNA (SEQ ID NO:22) as described in Example 7 as the starting template source, an additional 12 cycles of selection and amplification were performed with PCR mutagenesis preceding the first six selection cycles. It was assumed that mutagenesis of ligand A13 would yield ligands with a higher affinity for SP because the random sequence pool used to initiate Experiment A contained about 10¹⁴ unique sequences, only a minute fraction of the 4⁶⁰ possible sequences 60 nucleotides in length. Under these conditions, the evolution of a more favorable primary sequence solution to higher ordered structures would be essentially unavoidable. PCR products from the initial mutagenic PCR amplification of ligand A13 (RNA produced from this template pool was used for the first selection cycle) were cloned and sequenced to investigate the mutagenesis procedure. Ninety-six point mutations were identified within the 60N regions of 25 clones sequenced, representing a mutation rate of 0.064 per nucleotide position. At this rate, an average of 3.8 point mutations was expected per RNA per each of the first six selection cycles. Transition and transversion frequencies were equal (48 of each) , and the mutations appeared to be randomly distributed throughout the sequence space. However, as observed by Bartel and Szostak (Bartel, D. P., and Szostak, J. W. (1993) Science 261:1411-1418) . there was a bias in the types of mutations induced. With the non-mutated 6ON region of clone A13 having a nucleotide representation of 22 G, 20 C, 17 A, and 1 U, the following point mutations were identified: AT to TA (30) , AT to GC (22), CG to TA (14) , GC to AT (10), CG to AT (8) , GC to TA (4) , GC to CG (2) , AT to CG (2) , TA to CG (2), TA to AT (2), TA to GC (0), and CG to GC (0) . Selective pressure for the tightest binders was also increased in Experiment B by reducing the peptide concentration following cycles in which binding had significantly increased (Table 2) . The matrix-coupled SP concentrations were effectively lower than those listed in Table 2 (given as a function of column volume; 100 μl) , since binding occurred in a well-mixed 1-ml suspension. For selection cycles 8-12, a counterselection thiopropyl Sepharose matrix was used prior to SP affinity selection. An RNA pool generated from cycle 12 PCR products exhibited a dissociation constant of 0.80 μM for SP in an equilibrium dialysis binding experiment (Fig. 11) , a 7-fold improvement in binding over ligand A13. PCR products from the 12th selection cycle were cloned and sequenced. Of the 33 clones sequenced, all were unique (SEQ ID ΝOS:43-72) . However, all but three of the sequences can clearly be placed into two major sequence classes (Fig. 10) .

Binding of Ligands to SP in Solution. All 33 mutagenized clones were screened for their ability to bind SP in solution by single-point equilibrium dialysis measurements (Fig 10) . Ligands assigned to class 1 generally have the highest affinity for SP in solution, with ligand B28 (SEQ ID NO:53) having the lowest K_d (measured at 170 nM in this screen) . A five- point binding curve was subsequently performed by equilibrium dialysis (see Example 6) to obtain the more reliable ligand B28 K_d measurement of 190 nM (Fig. 11) . Experiment B, therefore, yielded ligands with binding affinities up to 30-fold better than their ancestral ligand A13 and approximately 6,000-fold better than the initial unselected random sequence RNA pool. With the exception of class 3 ligand B32 (SEQ ID NO:75) , all class 2 and class 3 ligands exhibited a K_d above 2 μM (Fig. 10) . A comparison of nucleotide positions 4-22 of the high affinity class 3 ligand B32 (SEQ ID NO:75) with positions 10-28 of class 1 ligands reveals a significant sequence similarity (GGC-NACCCUNAGG) (SEQ ID NO:79) , indicating the probable importance of this region in SP binding. The lower affinity class 3 ligands (B22 and B23) (SEQ ID NOS:74 and 73, respectively) share significant stretches of sequence homology with the relatively low affinity class 2 ligands: ACAGGACAC and GACGAGUU at positions 4-12 and 24-31 in the class 1 alignment, respectively (Fig. 10) .

EXAMPLE 9. Predicted Secondary Structure of Selected Ligands .

A comparative analysis of sequences within class 1 and 2 of Figure 10, with base-pairing decisions influenced by observed covariation, led to the prediction of possible secondary structures (Fig. 12) . This approach, known as the phylogenetic comparative approach, is a reliable method for determining secondary and tertiary structures of RNAs (and ssDNAs) (Fox, G. and Woese, C. (1975) Nature 256:505-507; Noller, H. F., and Woese, C. R. (1981) Science 212 :403- 410) . With this approach, one looks for structural features that are conserved despite differences in the primary sequence of the nucleic acids (i.e., covariance) . As an example, if an equivalent base- pairing scheme in a putative RNA helical region is not present in homologous regions of phylogenetically and functionally related RNAs, it is unlikely that it exists in the RNA's functional structure. Despite the high degree of sequence variability introduced during experiment B, a consensus structure predicted for the class 1 ligands resembles that predicted for A13 (SEQ ID NO:22) (Fig. 12) . The large number of selected mutations is not completely unexpected, as the random sequence RNA pool used to initiate experiment A contained about 10¹⁴ unique sequences, only a minute fraction of the 4⁶⁰ possible sequences 60 nucleotides in length. Under these conditions, the evolution of a more favorable primary sequence solution to higher ordered structures would be essentially unavoidable. Conserved nucleotides within the large asymmetric loop present in both the predicted ligand A13 (SEQ ID NO:22) structure and the class 1 consensus structure suggest a role for these nucleotides in binding. The majority of the nucleotides within this loop are completely conserved among class 1 ligands. In addition, the sequence within the high affinity class 3 ligand (B32) (SEQ ID NO:75) that is shared with the class 1 ligands (ACCCUNAGG) is present within this loop.

Experiment B class 2 ligands share a high degree of primary and secondary structure similarity; all can assume a stem-loop structure with two internal asymmetric loops (Fig. 12) . Most of the internal loop nucleotides are conserved, suggesting their involvement in the SP interaction. The nucleotides conserved among the lower affinity class 3 ligands (B22 and B23) (SEQ ID NOS:74 and 73, respectively) and the class 2 ligands (listed above) are partially present in the two internal loops and form the stem that separates them.

EXAMPLE 10.

Amino Acids Required for Interaction with Ligand B28. To determine which amino acids are required for the interaction with ligand B28 (SEQ ID NO:53), overlapping fragments of the peptide were tested for their ability to compete with intact SP (SEQ ID NO:23) for binding in equilibrium dialysis experiments, as described above. Each competition experiment contained 1.6 μM B28 RNA and 1.6 μM SP (P₀; including 20 nM ³H-

SP) , in addition to 32 μM (P₀) of the competing peptide fragment (20:1 ratio of competing peptide to SP) . In the absence of competing peptide, 63% of the SP was bound under these conditions. The percent inhibition observed with 32 μM competing unlabeled SP (as a positive control) was 86%. The results (Fig. 13) indicate that the four C-terminal residues (Phe^β-Gly⁹- Leu^-Met^-NH.) (SEQ ID NO:80) are not necessary for binding. The presence of Phe⁷ appears to be required for optimal binding, but is not entirely necessary.

The involvement of Arg¹ in the interaction is indicated by the inability of fragment 2-11 to compete under these conditions. However, the four N-terminal residues (Arg¹-Pro²-Lys³-Pro") (SEQ ID NO: 81) alone did not exhibit significant binding, suggesting a requirement for one or both of the Gin residues . Taken together, the results suggest that, minimally, Arg¹ and Gln⁵-Gln⁶-Phe⁷ are involved in the interaction with ligand B28 (SEQ ID NO:53) .

EXAMPLE 11. Specificity of ligand B28 for SP over rSP. A high specificity of ligand B28 (SEQ ID NO:53) for SP was suggested by the ability of the ligand to discriminate between SP and the reverse orientation of the same peptide (rSP, Fig. 8) . An equilibrium dialysis competition experiment was performed as above, except the competing concentrations (P₀) of rSP were either 1.6, 6.4, 25, or 100 μM. Competition experiments with unlabeled SP at the same concentrations were performed for comparison. In the absence of added competitor, 69% binding of SP was observed under these conditions. The data indicate that rSP is a poor competitor for binding to ligand B28 (SEQ ID NO:53) (Fig. 14) . Knowing that multiple amino acid residues are involved in the interaction (Fig. 13) , it can be assumed that SP possesses a unique structure, with unique relative positions of amino acid side chains, as it is recognized by the ligand. Although this recognized structure might include the N-terminal arginine alpha- amine of free SP, the N-terminus of the column-coupled peptide (Cys-SP, Fig. 8) , to which ligands were selected, was acetylated. Chassaing et al . (1986) Eur. J. Biochem. 1 4:77-85 have proposed a preferred conformation of SP in methanol consisting of a flexible Arg¹-Pro²-Lys³ N-terminus, and alpha-helical structure involving residues Pro⁴-Gln⁵-Gln⁶-Phe⁷-Phe⁸ (SEQ ID

NO:82) and an interaction of the C-terminal carboxamide with the primary amides from both glutamines. An alpha-helical structure may not exist in the core residues of rSP, for instance, because these residues would not have the benefit of the helix-nucleating properties of proline (Presta, L. G., and Rose, G. D. (1988) Science 240:1632-1641) .

EXAMPLE 12.

Modified 2 ' -NH. Pyrimidine RNA Ligands to SP. In order to generate ligands with improved stability in vivo, an experiment is carried out with randomized RNA containing amino (NH₂) functionalities at the 2'- position of each pyrimidine. A library of 10¹⁴ RNA molecules is generated that contains 60 nucleotides of contiguous random sequence flanked by defined sequences. Defined nucleotide sequences in the flanking regions of the template serve as primer annealing sites for PCR and also provide the promoter sequence ( e . g. , Tl ) required for transcription. The random nucleotides of the initial candidate mixture are comprised of 2'-NH₂ pyrimidine nucleosides. The rounds of selection, amplification, and optionally mutagenesis are carried out as described in Examples 6-8 using art- known techniques.

To avoid the selection and amplification of undesired RNA molecules that bind to matrix components other than the target peptide, counterselection steps preceding the selection steps are incorporated into the selection process (as described above) . These steps are reiterated until the enriched pool of RNA shows significantly improved affinity to SP over the initial random pool. The resulting RNA ligands are reverse transcribed and the cDNAs are PCR amplified. The dsDNA PCR products are cloned into a plasmid vector and sequenced as described above. EXAMPLE 13. EXPERIMENTAL PROCEDURES

This Example provides general procedures followed and incorporated into Examples 14-19.

Materials . HIV-1 integrase, isolated from BHIO, was a generous gift from Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, CA 92121-1121. The BHIO clone is also publicly available from the AIDS Reagent Program, 685 Lofstrand Lane, Rockville, Maryland 20850. Isolating and purifing HIV-1 integrase from BHIO would be routine for those skilled in the art. In addition, integrase (IIIB) can be purchased from Intracel Corporation, 359 Allston Street, Cambridge, MA 02139. DNA polymerase was purchased from Perkin Elmer Cetus. Alkaline Phosphatase (Calf Intestinal) was purchased from

Biolabs. T4 polynucleotide kinase was purchased from Boehringer. Cobra Venom Ribonuclease (V_x) was purchased from Pharmacia, and Ribonuclease T_x was purchased from Boehringer. All other enzymes were purchased from commercial sources. pUC18 was purchased from BRL.

PCR Amplification and Selection. SELEX was carried out essentially as described in the SELEX Patent Applications (see also Tuerk and Gold (1990) Science 249:505-510) . A random pool of DNA 10¹⁴ oligomers was synthesized where the 5' and 3 " proximal ends were fixed sequences used for amplification and the central region consisted of thirty randomized positions. (See Table 3 for the starting ssDNA template (SEQ ID NO:140) , the 3' PCR primer (SEQ ID NO:141) , and the 5' PCR primer (SEQ ID NO:142) Ten picomoles of template were PCR amplified for 8 cycles in the initial round. Copy DNA of the selected pool of RNA from subsequent rounds of SELEX was PCR amplified 18 cycles. PCR reactions were carried out in 50 μl volume containing 200 picomoles of each primer, 2mM final concentration dNTP's, 5 units of Thermus aquaticus DNA polymerase (Perkin Elmer Cetus) in a PCR buffer (10 mM Tris-HCl pH 8.4, 50 mM KCl, 7.5 mM MgCl₂, 0.05 mg/ml BSA) . Primers were annealed at 58 °C for 20 seconds and extended at 74 °C for 2 minutes. Denaturation occurred at 93"C for 30 seconds.

Products from PCR amplification were used for T7 in vi tro transcription in a 200 μl reaction volume (Tuerk and Gold (1990) Science 249:505-510) . T7 transcripts were purified from an 8 percent, 7M Urea polyacrylamide gel and eluted by crushing gel pieces in a Sodium Acetate/EDTA solution. For each round of SELEX, 50 picomoles of the selected pool of RNA was phosphatased for 30 minutes using Alkaline Phosphatase, Calf Intestinal (Biolabs) . The reaction was then phenol extracted 3 times and chloroform extracted once, then ethanol precipitated. 25 picomoles of this RNA was 5' end-labeled using γ-³²P ATP with T4 polynucleotide kinase (Boehringer) for 30 minutes. Kinased RNA was gel purified and a small quantity (about 150 fmoles; 100,000 cpm) was used along with 250 picomoles of cold RNA to follow the fraction of RNA bound to integrase and retained on nitrocellulose filters during the separation step of SELEX. Typically a protein concentration was used that binds one to five percent of the total input RNA. A control (minus protein) was used to determine the background which is typically ≤ 0.1% of the total input. Selected RNA was eluted from filter by extracting three times with H₂0 saturated phenol containing 2% lauryl sulfate (SDS) , 0.3 M NaOAc and 5 mM EDTA followed by a chloroform extraction. Twenty five percent of this RNA was then used to synthesize cDNA for PCR amplification.

Selection with non-amplifiable Competitor RNA. Selections were done using two buffer conditions where the only difference between the buffers is sodium concentration (250 mM NaCl or 500 mM NaCl) . Two different buffer conditions were used to increase stringency (with the higher salt concentration being more stringent) and to determine whether different ligands would be obtained. After 10 rounds of SELEX, the binding constant of the selected pool decreased about an order of magnitude and remained constant for the next two additional rounds. Competitor RNA was not used in the first 12 rounds. After this round, the pool was split and selection was carried out in the presence and absence (control) of competitor RNA. For rounds 12 through 18, a 50-fold excess of a non- amplifiable random pool of RNA was present during selection to compete with non-specific low-affinity binders that may survive and thus be amplified. The competitor RNA, which had a 3ON random region, was made as described supra for the amplifiable pool RNA; however, the competitor RNA had different primer annealing sequences (3' PCR primer, RNA reverse transcription primer: CCCGGATCCTCTTTACCTCTGTGTG (SEQ ID NO:143) ; 5' PCR primer, T7 promoter:

CCGAAGCTTAATACGACTCACTATAGGGACTATTGATGGCCTTCCGACC (SEQ ID NO:144) . Thus, the competitor RNA does not survive the cDNA synthesis or PCR amplification steps. It would be apparent to one skilled in the art that other primer sequences could be used as long as they were not homologous to those used for the pool RNA. The use of competitor RNA increased the affinity of the selected pool by several orders of magnitude. In addition, RNA sequencing of the selected pool after using competitor RNA in three rounds of selection (round 15 of SELEX) showed non-randomness in the sequence, whereas the pool of RNA that survived SELEX (round 15) with no competitor still appeared random. Control experiments where competitor RNA was used in the absence of selectable RNA produced no PCR product after 35 cycles of PCR amplification.

Cloning and Sequencing. PCR amplified DNA from the round 18 selected-pool of RNA was phenol and chloroform extracted and ethanol precipitated. The extracted PCR DNA was digested using Bam HI and Hind III (Biolabs) and subcloned into pUC18. DNAs were phenol and chloroform extracted following digestion. Ligation was carried out at room temperature for two hours after which time the reaction was phenol and chloroform extracted and used to electroporate competent cells. Fifty transformants from the selections using competitor RNA at both NaCl concentrations were picked and their DNAs sequenced. Chemical and Enzymatic Structure Probin . RNAs were chemically modified using DMS (dimethyl sulfate) , kethoxal (2-keto, 3-ethoxy-n-butryaldehyde) and CMCT (l-Cyclohexyl-3- (2-Morpholinoethyl) -Carbodiimide Metho- p-Toluene-sulfonate) and partially digested using Cobra Venom Ribonuclease (V₁ ; Pharmacia) and Ribonuclease T-_. (Boehringer) as described (Allen and Noller (1989) J. Mol. Biol. 203 :457-468) with the following exceptions. Each modification reaction contains one of the following reagents, 2 μl of a 1:15 dilution of DMS in 100% ethanol; 4 μl of kethoxal at a concentration of 25 μg/μl in 50% ethanol; 25 μl of CMCT at 40 μg/μl in CMCT modification buffer (80 mM potassium borate (pH 8.0) , 10 mM MgCl₂, 100 mM NH₄C1) ; 2 μl of 0.01 unit/μl RNase Ti and 2 μl of .001 unit/μl RNase V-_.. Prior to modification, RNAs were incubated at 45^* C for 5 minutes in DMS and kethoxal modification buffer (80 mM potassium cacodylate (pH 8.0) , 10 mM MgCl₂, 100 mM NH₄C1) or CMCT modification buffer. Samples were then incubated for 20 minutes at 37^* C in the presence or absence of integrase protein. Chemical modifications were carried out in a reaction volume of 50 μl and were done at 37^* C for 8 minutes and enzymatic digestion for 3 minutes at the same volume and temperature. Each reaction contained 10 picomoles of RNA (0.25 μg) . In reactions containing integrase, the integrase concentrations were 5 x 10^"7 M or 1 x 10^"s M. RNAs that were digested with ribonucleases were done only in the absence of integrase. Modified RNAs were then phenol extracted twice and chloroform extracted once and primer extended (Stern et al . (1988) Meth. Enzymol. 164 :481-489) to determine positions that were accessible to the probes. The positions are identified by a pause or stop by reverse transcriptase.

Binding Assays . Binding assays were done by adding 5 μl of HIV-1 integrase protein, at the appropriate concentrations (i.e., ranging from 2 X 10^"6 with 3 fold dilutions to 9 X 10^'9 for 250 mM NaCl and 0.5 X 10^"7 with 3 fold dilutions to 2 X 10^"10 for 50 mM NaCl) , to 45 μl of binding buffer (50 mM Na-HEPES pH 7.5, 250 mM NaCl, 2 mM DTT, 10 mM MnCl₂, 5 mM CHAPS) on ice, then adding 50,000 cpm of kinased RNA (<200 fmoles) in a volume of 3 to 4 μl. This mix is incubated at 37^*C for 20 minutes. The reactions were then passed over nitrocellulose filters, which were pre-equilibrated in buffer, and washed with a 50 mM Tris-HCl pH 7.5 solution. Filters were dried and counted in cocktail. The proteins used in these experiments were frozen and thawed only once. Each binding curve consisted of seven points and each point is an average since experiments were done in triplicate.

In Vi tro Processing Assay. Con(+) (5' - CAATGACCGCATGGGATCCGTGTGGAAAATCTCTAGCAGT-3') (SEQ ID NO: 139) and Con(-) (5' - ACTGCTAGAGATTTTCCACACGGATCCCATGCGGTCATTG-3') (the complement of SEQ ID NO:139) were the two DNA oligomers used to mimic the U5 region of the HIV-1 genome. Con(+) was end-labeled and annealed to Con(-) and the duplex purified on an 8% native acrylamide gel. The two strands were annealed by adding 2.5 fold excess of Con(-) strand to the Con(+) kinase reaction on ice and then heated immediately to 90°C for 3 minutes and allowed to cool slowly to 40°C then loaded on to gel. Purified duplex DNA was resuspended at 0.65 pmol/μl concentration. In a 10 μl reaction volume, 0.13 picomole of duplex DNA was incubated with integrase at 0.4 x 10^"6 M in reaction buffer (50 mM Na-HEPES pH 7.0, 50 mM NaCl, 2 mM DTT, 2.5 mM MgCl₂) for 20 minutes at 37°C. When inhibitor RNA was present in reaction, the concentration was 600 nM or lower. Similar methods have been used to assay this activity (Sherman and Fyfe (1990) Proc. Natl. Acad. Sci. USA £7:5119-5123; Bushman and Craigie (1991) Proc. Natl. Acad. Sci. USA 88 :1339- 1343)

Competition Assay. Radioactively labeled con(+) oligo was annealed to con(-) and purified on a 8% denaturing gel. 50 nM integrase was mixed with 20 nM double-stranded con+/- which mimics the U5 region of the HIV genome and incubated at 37°C for 5 minutes. After this time, varying amounts of P5 or 3ON RNA (i.e., random pool RNA) was added and the reaction mix (30 μl final volume) was incubated further for 20 minutes. The reactions were then placed at room temperature and passed over nitrocellulose filters. Filters were washed three times with 1 ml of 50 mM Tris-HCl pH 7.5. Filters were dried under a heat lamp and counted in a cocktail for 1 minute each. In the absence of RNA under these conditions, approximately 10% of the double-stranded DNA was retained on filter. The buffer used in these assays was the same as the selection buffer except that the sodium chloride concentration was 180 mM.

EXAMPLE 14.

Sequence Analysis. After 18 rounds of SELEX, individual RNA molecules were isolated with increased specificity over the parent pool. Sequences cloned from round 18 selected pool fell into three major groups. The group with the highest affinity to integrase (group I) has 36 members. Eighteen clones in this group were identical and this sequence (P5) was found to be the best binder (Table 4) . There are three RNA molecules in this family with a single base substitution (A15) and 2 with two bases substituted (P29) . The other 13 members of group I contain 3 or more base changes (P54, P23, AND PI) . There was a correlation between sequence similarity to the predominant sequence (designated P5) and affinity for integrase. Though sequence changes in general appear to preserve the structure, RNAs with fewer changes away from P5 bound better. The second group of molecules (group II) bound with an affinity which was significantly less than the group I RNAs. There are 20 members in group II of which, eight are the consensus (designated A54) , four contain a single base change (P56) , and the rest contain 3 or more changes (A54, P47, P64, and All) . The third grouping of sequences (group III) contains sequences which have little or no homology. However, some sequences share short sequence motifs. In addition, all of the members of group III that were tested bound significantly better than the parent pool even though binding was considerably lower than the other two groups. In general, the SELEX done at high NaCl concentration resulted in a greater number of sequences belonging to the group I class of molecules, while the SELEX experiment done at low salt gave more RNAs belonging to the group II class, which is consistent with the results from sequencing the selected pools of RNA (Table 4) .

Filter Binding Studies. Binding curves were generated for 20 different individual RNA species chosen randomly between the groups. These experiments were carried out in triplicate. All binding curves were done using γ-³²P labeled RNA in binding buffer containing 250 mM NaCl. (K_d was figured from the equation Y=M₀/ (M₂ + M₀) X M_lf where M₀ = X-axis concentration value, M-_. = maximal Y value, M₂ = K_d, and Y= % RNA bound) . The binding affinities correlate directly with the size of the groups. The highest affinity molecules are from group I, and the best binder of that group is the RΝA most frequently represented, P5. The dissociation constant for P5 RΝA is on the order of 12 x 10^~9 M in binding buffer containing 250 mM aCl. P23 has a K_d of 25 X 10^~9 M in binding buffer containing 250 mM ΝaCl. The K_d for the parent pool of RΝA under these same conditions is -15 x 10^"6 M. 21-mer DΝA oligos with identical sequence as the U5 terminal region of HIV-1 genome were made and used to generate binding curves. Both single-stranded and double-stranded U5 DΝA bound with dissociation constants greater than 20 x 10^"6 (data not shown) . When binding reactions were carried out in binding buffer containing 50 mM ΝaCl, all the dissociation constants decreased as expected. The K_d of P5 RΝA in 50 mM ΝaCl decreased to 2 x 10^"9 M while the K_d for 30Ν pool of RNA improved to 2.5 x 10^"8 M. In binding buffer containing 50 mM NaCl, U5 DNA oligos bound with about the same K_d as 3ON RNA.

The dissociation constant for the group II RNAs is on the order of 80 x 10^"9 M in buffer containing 250 mM NaCl. In particular, A63 had a dissociation constant of 125 X 10^"9 M in 250 mM NaCl. RNAs taken from the third group had K_d values of approximately 8 x 10^"7 M. (Al, A2, A42, and A47 had Kd values ranging between 800 and 1000 nM) . Although group III molecules do not show significant relatedness, all of the members that were tested bound better than the initial 3ON pool.

EXAMPLE 15. PREDICTED SECONDARY STRUCTURE OF

SELECTED LIGANDS Computer Analysis of RNA Structure. RNAs were folded using the Zucker folding program. Structures were taken with calculated folding energies ≤-10.5 kcal/mol. In general, related RNAs that were grouped were able to adopt similar secondary structures. The structure for P5 molecule can be formed by nearly all the members of group I (SEQ ID NO: 84; Fig. 15A) . The computer-derived structure for group I RNAs appears to be quite stable. However, there are several non- canonical base pairing interactions; G18 with U52, U37 with G46 and U39 with G44 which is at the end of a stable GNRA tetra-loop. In addition, there seems to be higher-order interactions between nucleotides within loop 10 and loop 30 (Fig. 15A) . The interactions between these two loops include the final non-Watson- Crick base pairing between bases U13 and C29. Although this interaction is not as favorable as G-U pairing, evidence for C-U pairing in 5S Ribosomal RNA does exist (Wu and Marshall (1990) Biochem. 29_ : 1730-1736) . Since the nucleotides in loop 10 are part of the fixed sequence used for PCR amplification, there are no phylogenetic data to support the proposed interaction between the loops. The computer model for P5 folding is reasonable. Calculated free energy for this structure is -12.8 kcal/mole and other sequences with mutations away from the primary sequence can fold up with the same structure. In addition, this model is in good agreement with chemical modification data ( infra) . There is some ambiguity in the folding of nucleotides 36 to 48. It is probable that the nucleotides at these positions are involved in alternative structures (Fig. 15A) . The most likely computer model for group II RNAs is the stable structure calculated for A54 at -16.6 kcal/mole (SEQ ID NO: 90; Fig. 15B) . The variants in this group are able to fold up into this structure. A54 has a few features that are interesting. The nucleotides at the 5' and 3' ends are single-stranded. There is a large (11 bases) purine-rich loop near the 5' end-. The stems in this structure are all very G/C rich. There are four non-canonical base pairs and they are all G-U pairs . A consensus structure could not be found for members of group III.

Chemical and Enzymatic Structure Analysis . The structure deduced for P5 RNA modified in the absence of integrase with structure-specific chemical probes and partially digested with RNases supports the computer model. Positions that were modeled as single-stranded were accessible to DMS, kethoxal and CMCT. RNase V-_. only showed cuts in the stem formed by nucleotides 15 to 21 and 49 to 55 (data not shown) . This lends support to the computer structure since V-_. preferentially cleaves double-stranded RNA of length 5 base pairs or greater (Lockard and Kumar (1981) Nucleic Acids Res. :5125-5140; Favorova et al . (1981) Biochem. 20:1006-1011) . Nucleotides 10 to 14 and 27 to 32 are modeled as being single stranded. However, the nucleotides in these loops are relatively unreactive to the modifying reagents. In addition, a putative pseudoknot can be formed between nucleotides 11 to 14 and 28 to 31. Nucleotides G10 and G32, which are single-stranded, show normal reactivity to kethoxal while G27 (also single-stranded) appears significantly less accessible. This may be a result of the interaction between the two loops. Nucleotide A43 which is modeled as being single stranded is also relatively unreactive. This base may be buried by the structure of the GNRA tetra-loop. It was not possible to study the reactivity of nucleotides 1 to 3 and 65 to 77, since we used a primer 10 bases long to anneal to the 3' end and the terminal transcript signal masks the three G's at the 5' end.

A number of positions that were accessible to modifying reagents in the absence of integrase were shielded when modification was carried out in the presence of integrase (integrase was present at concentrations of 5 x 10^~7 M and 1 x 10^"6 M) . Nucleotides A22, A23, G27, G34, A41, G46, G48, G49 and G53 were all completely protected from attack by chemicals at 5 x 10^"7 M integrase concentration. Positions G40 and G44 were partially protected at 5 x 10^"7 M and showed further protection at 1 x 10^"6 M.

Protection by integrase can be interpreted as either direct shielding by the protein or structural perturbation of the RNA upon binding the protein. It appears the protection of residues G34, G44, G46, G49 and G53 is caused by stabilization of the RNA structure by the binding of integrase. Nucleotide A43 was minimally reactive to DMS and showed no protection by integrase, whereas the other bases in this tetra-loop were highly reactive and were strongly protected when integrase bound. It has been shown that nucleotides in GNRA tetra-loops in 16S ribosomal RNA have chemical modification patterns that are different from those of single-stranded nucleotides (Moazed et al . (1986) J. Mol. Biol. 187 :399-416) . In other words, whereas bases in single-stranded regions are accessible to chemical probes, tetra-loop nucleotides may form structures such that they are protected (Chastain and Tinoco (1991) Prog. Nucl. Acid Res. and Molec. Bio. 41 :131) .

EXAMPLE 16.

RNA-truncate Studies. Truncates of P5 RNA were made to determine the minimal binding domain of the RNA. Truncated RNAs were designed based on the results from chemical protection studies. Deletion of nucleotides 1 to 6 and 64 to 77 had no noticeable effect on binding of the RNA to integrase. In addition, changing the sequence in the 3 base stem, of this truncate, which holds the 5' and 3' ends together does not affect binding. However, deleting either nucleotides 1 to 14 or 56 to 77 completely abolished binding. This result argues that there may be an interaction between the nucleotides in the loop around position 12 and position 30 that is important for binding. However, this interaction alone is not sufficient, as a stem which holds the 5' and 3' ends together is necessary. The only other argument for this interaction is that the UCUU sequence is highly conserved in the group I class of molecules even though the random region begins at position A23 and ends at G53. There is good agreement between the chemical protection data and the minimal binding domain, as all the nucleotides that are protected by integrase are within the structure that binds with the same K_d as the mature molecule.

EXAMPLE 17. In Vi tro Inhibition of Integrase. When integrase was added to a reaction containing double-stranded DNA that mimics the U5 (or U3) region of HIV DNA, two nucleotides were removed from the 3' end of the strand which encodes the conserved CA near the 3' end. In a buffer containing 50 mM NaCl, about 50% of the 40 nucleotide end-labeled strand was converted to a 38 nucleotide fragment. This activity was completely inhibited by P5 at sub-micromolar concentrations (200 nM or less) . Since the difference in affinity between P5 and random RNA at 50 mM NaCl was only a few fold, as expected there was no significant difference in inhibition observed with P5 and 3ON at this salt concentration. Similar results were obtained at a NaCl concentration of 75mM. P5 was selected from a random pool of 3ON molecules at 250 mM NaCl. Processing activity at NaCl concentrations above 100 mM is greatly diminished.

EXAMPLE 18. Competitive Binding of P5 RNA. Since integrase showed no processing activity at the sodium chloride concentrations at which the selections were done (i.e., 250 mM and 500 mM NaCl) , competitive binding studies were carried out at a salt concentration that allows integrase to discriminate between specific and random RNAs. In binding buffer containing 180 mM NaCl, integrase was able to bind the same substrate (double- stranded con+/- DNA; U5 mimic) that it is able to process at 50 mM NaCl. In the 180 mM NaCl buffer, P5 inhibits 50% of the substrate binding at a concentration of 6 nM, while it took approximately 500 nM 3ON RNA to provide the same extent of inhibition.

Although we were not able to demonstrate directly that P5 inhibits the processing activity of HIV integrase, it is reasonable to conclude that if integrase was functional in vi tro at these salt concentrations, P5 would be a potent inhibitor. Since the Ki for P5 is similar to the K_d, this suggests that P5 competes directly for binding with DNA substrate. Therefore, it is reasonable to conclude that under in vivo conditions where the ionic strength is much greater, P5 binds integrase with high affinity and specificity.

Blockage of integrase activities should.prove to be a potent inhibitor of viral production. Similar experiments were done using HIV-1 reverse transcriptase (RT) (Tuerk et al . (1992) Proc. Natl. Acad. Sci. USA £9:6988-6992) . Tuerk and Gold showed that RNAs derived from SELEX to bind RT were strong inhibitors of reverse transcription (Tuerk et al . (1992) Proc. Natl. Acad. Sci. USA £9=6988-6992) and RNase H (Chen and Gold (1994) Biochem. 3_3_:8746-8756) activities in vi tro . Thus, it is reasonable to conclude that these RNAs would have the same effect when over-expressed in vivo . One important advantage of having high affinity nucleic acid inhibitors is that these act as competitive inhibitors. Since these RNA ligands bind at the same site as DNA substrates, protein mutations that reduce the affinity of the inhibitor may also reduce the affinity of the substrate. However, the size of the RNA ligands (i.e., large relative to other inhibitors) makes them less likely to encounter mutations that confer resistance to these inhibitors, which is a major problem with the therapeutics used to combat HIV today (Larder et al . (1989) Science 211:1731-1734) .

EXAMPLE 19.

Modified 2' -NH-, Pyrimidine RNA Ligands to HIV-1 Integrase. In order to generate ligands with improved stability in vivo, an experiment is carried out with randomized RNA containing amino (NH₂) functionalities at the 2' -position of each pyrimidine. A library of 10¹⁴ RNA molecules is generated that contains 30 nucleotides of contiguous random sequence flanked by defined sequences. Defined nucleotide sequences in the flanking regions of the template serve as primer annealing sites for PCR and the complement of the primer provides the T7 promoter sequence (a restriction site can be added for cloning) . The random nucleotides of the initial candidate mixture are comprised of 2'- NH₂ pyrimidine bases. The rounds of selection, and amplification are carried out as described supra in Examples 13-14 using art-known techniques.

EXAMPLE 20. EXPERIMENTAL PROCEDURES

This Example provides general procedures followed and incorporated into Examples 21-25.

Materials. Recombinant HIV-1 RT overexpressed in E. coli cells was purified according to the procedure described in Davies, J. F. et al . (1991) Science

252 :88-95. Enzyme was aliquoted and stored at -70°C in HRT Buffer (200 mM KOAc, 50 mM Tris-Acetate, pH 7.4, 6 mM MgCl₂, 10 mM DTT) . Aliquots thawed and refrozen more than once were discarded. All other materials were purchased from commercial sources.

Generation of Degenerate ssDNA Library. A population of synthetic DNA oligonucleotides (oligo 1) (SEQ ID NO:145) containing 35 random nucleotides flanked by invariant primer annealing sites was amplified by the Polymerase Chain Reaction (PCR) using oligos 2 (SEQ ID NO:146) and 3 (SEQ ID NO:147) as primers (Fig. 16) . Oligo 3 (SEQ ID NO:147) had three biotin phosphoramidites covalently attached to its 5' terminus during synthesis. The 81 nucleotide double- stranded PCR product was size-purified on a 12% non- denaturing acrylamide gel and 100-300 pmol were applied to 100 μl of a Pierce streptavidin-agarose bead matrix suspended in Buffer A (50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA) . After equilibration for 30 minutes at 20°C to allow the biotinylated double-stranded DNA (dsDNA) to bind the streptavidin beads, unbound dsDNA was removed with five 500 μl washes of Buffer A, and the matrix-bound dsDNA was denatured in 400 μl of 0.15N NaOH for 15 minutes at 37°C. As these conditions were not harsh enough to disrupt the biotin-streptavidin interaction, denaturation released only the non- biotinylated DNA strand from the bead complex. The free DNA was collected and precipitated, yielding 70- 200 pmol of single-stranded DNA (ssDNA) . 10-20 pmol were ³²P labeled at the 5' end with T4 Polynucleotide Kinase and the product was size-purified on an 8% denaturing acrylamide gel and combined with the remaining (unlabeled) ssDNA to comprise the degenerate ssDNA library used for the selections.

Nitrocellulose Filter Binding Assays. Oligonucleotides bound to proteins can be effectively separated from the unbound species by filtration through nitrocellulose membrane filters (Yarus, M. and Berg, P. (1970) Anal. Biochem. £5:450-465; Lowary, P.T. and Uhlenbeck, O.C. (1987) Nucleic Acids Res. 15:10483- 10493; Tuerk, C. and Gold, L. (1990) Science 24_.9:505- 510) . The affinity of the random ssDNA library for HIV-1 RT was determined using a protein excess nitrocellulose filter binding assay as described in Carey, J. et al . (1983) Biochemistry 2_2:2601-2609. Selections were performed with a saturating ssDNA concentration to promote competition among DNA ligands for a limited number of available target binding sites . The percent of target-dependent DNA retention was minimized for each selection to ensure maximum enrichment of the library for target binders; however, to avoid propagation of members with high affinity for nitrocellulose, selections were repeated if target-free (background) retention was greater than 10% of target- dependent retention.

For the first selection, 500 nM HIV-1 RT and 2 μM ssDNA (100 pmol or about 10¹⁴ different molecules) were equilibrated at 37°C for 5 minutes in HRT Buffer and filtered through nitrocellulose to sequester target- bound ligands. Target-free selections were done in duplicate to measure and correct for background binding levels. The fraction of total DNA retained by the filters was calculated by measuring radiation without fluor in a scintillation counter. Ligands were harvested from the filter as described in Tuerk, C. and Gold, L. (1990) Science 2.9-505-510, amplified by PCR, denatured from the biotinylated complementary strand, and end-labeled as described above to regenerate the library. The affinity of the pool for HIV-1 RT was measured prior to selections 6, 8, 10 and 12, and was estimated for the remaining selections. These values determined the ligand concentration necessary for saturation each selection. As the affinity of the population for HIV-1 RT increased, the concentrations of ligand and RT were reduced accordingly to increase selection stringency.

Equilibrium Dissociation Constants. In the simplest case, equilibrium binding of ssDNA ligand (L) to HIV-1 RT protein (P) can be described by equation (1) =

PL •* P_f + L_f (1)

where K_d = ( [P_f] [L_f] / [PL] ) is the equilibrium dissociation constant between the protein and ssDNA ligand, P_f is free protein, L_f is free ligand, and PL is protein-ligand complex. Using the mass balance equations, the fraction of bound ligand at equilibrium (q) can be expressed in terms of measurable quantities according to equation (2) :

q = (P_t + L_t + K_d - ((P_t + L_t + K_d) ² - 4 P_tL_t)"=) (2)

where P_t and L_t are total protein and ligand concentrations .

For competition experiments an additional equilibrium exists between the protein (P) and competitor (C) as described by equation (3) :

K_c PC »- P_f + C_f (3)

where K_c = ( [P_£] [C_f] / [PC] ) is the equilibrium dissociation constant between the protein and competitor, P_f is free protein, C_£ is free competitor, and PC is protein-competitor complex. Competition titration experiments were analyzed using equation (4) to determine the concentration of free protein as a function of the total competitor concentration:

[P_t] = [P_f] ( 1 + K_d [L_t] / ( 1 + K_d [P_f] ) + K_c [C_t] / ( 1 + K_c [P_f] ) ) ( 4 )

This equation assumes a 1:1 binding stoichiometry for both reactions and that only one species is bound to protein at a time. Since it is difficult to obtain a direct solution for this equation in terms of [P_f] , we have utilized iteration to determine values of [P_£] to a precision of 1 x 10". To utilize this equation to follow [PL] as a function of competitor added, we also need the following expression:

[PL] = K_d [P_f] ([L_t] / (1 + K_d [P_f])) (5)

These equations were used in the non-linear least- squares data analysis to obtain the best fit parameters for K_c as a function of [C_t] for each competition experiment. The value used for K_d for this fitting analysis was the mean experimental value determined with equation (2) in the absence of competitor. Inhibition titration experiments were also analyzed using equations (4) and (5) , with the primer:template junction substrate as the ligand (L) and the ssDNA ligand the competitor (C) . Inhibition values are reported as K_A' rather than Ki (traditionally measured using a Michaelis-Menten analysis comparing reaction rates as a function of substrate concentration) because their mode of inhibition is likely a binding competition between substrate and ssDNA ligand, more accurately described by a K_c value as illustrated above.

Cloning and Sequencing Isolates. Following round 15, one pmol of the library was amplified by PCR using oligo 3 (SEQ ID NO:147) without the biotins (containing a Pst I restriction endonuclease cleavage site) and oligo 4 (SEQ ID NO:148) (containing a Bam HI site) as primers (see Fig. 16) . Double-stranded products were digested with Pst I and Bam HI and subsequently ligated into pUC19, similarly digested prior to the ligation.

The vectors were electroporated into E. coli DHlα cells and oligo 5 (SEQ ID NO:149) , complementary to 16 nucleotides of the PUC19 polylinker region, was used as a primer for dideoxy sequencing of the cloned inserts. These techniques are well-known in the art. A detailed description of these techniques can be found in Schneider, D. et al . (1993) FASEB 2=201-207. Large quantities of individual DNA ligands were prepared by amplifying the vector inserts by PCR using oligos 2 (SEQ ID NO:146) and 3 (SEQ ID NO:147) as primers and following the streptavidin matrix purification technique described above to isolate ssDNA.

Assay For Inhibition of RNA-Dependent DNA Polymerase Activity. A substrate for the RNA-dependent DNA polymerase activity of HIV-1 RT was assembled by annealing an 18 nucleotide, 5' end-labeled DNA primer to a 30 nucleotide RNA template with a complementary 3' end (see Fig. 25A) , and purifying the duplex on a 12% non-denaturing acrylamide gel. The primer sequence matched the 3' terminal 18 nucleotides of tRNA^lys'³, responsible for priming minus-strand DNA synthesis of the HIV-1 genome, and the template sequence paralleled the HIV-1 genomic primer binding site and downstream 12 nucleotides. A dilution series of inhibitory ssDNA ligand (to give a final concentration of 0, 1, 3, 9, 27, or 81 nM) was denatured in HRT Buffer at 70°C for 5 minutes and allowed to renature slowly at 20°C. The primer:template substrate was added to a final concentration of 40 nM, along with dNTP's at 400 μM. The 10 μl reaction was initiated with the addition of either HIV-1, AMV, or MMLV-RT (to give a final concentration of 10 nM) , allowed to proceed for 5 minutes at 37°C, and terminated with one volume of formamide. Extension products were separated on an 8% denaturing acrylamide gel and quantitated with an Ambis radioanalytic imager. Intramolecular Extension Assay. In a 10 μl reaction, 0.1 pmol of 5' end-labeled ssDNA ligand was denatured and slowly renatured as above and combined with 400 μM dNTP's and a saturating concentration of enzyme (200 nM HIV-1 RT, 100 nM AMV RT, or 0.6 units/ml Sequenase T7 DNA polymerase, all shown to have equivalent activity) , extended for 30 minutes at 37°C, and terminated with one volume of formamide. To determine the precise location of the annealed 3' end, extensions were also done with Sequenase in the presence of 25 μM ddATP. Extension products were separated on an 8% denaturing acrylamide gel. Biased Randomization Selections. A library of ligand RTl variants was chemically synthesized, incorporating the "wild-type" nucleotide at a frequency of 0.625 and each of the "mutant" nucleotides at a frequency of 0.125 in the 35N cassette. Selections for HIV-1 RT affinity were performed as described above; however, a simpler protocol was used to isolate and label the non-biotinylated DNA strand. During the amplification step, α-³²P dATP was incorporated into both strands of the duplex. The strands were separated on an 8% denaturing acrylamide gel by virtue of the retarded migration of the strand possessing the three biotins, and the non-biotinylated strand was recovered. Because the ssDNA was internally-labeled, end-labeling was not necessary and the recovered sample was ready for the next selection round.

EXAMPLE 21. DNA LIGANDS TO HIV-1 RT

Selected ssDNA Ligands Bind 700 Times Better After 15 SELEX Cycles. Following the selection guidelines described in Example 20, we were able to enrich the DNA library for RT binders from an initial apparent K_d value of 1400 nM to a final value of 4 nM in 12 cycles (see Fig. 17) . Enrichment began slowly, requiring 8 cycles to improve the affinity by one log (the apparent K_d of the round 8 library was 150 nM) , but increased quickly in the later cycles as predicted by Irvine, D. et al. (1991) J. Mol. Biol. 221:739-761, with the affinity improving another 10-fold by round 10 (K_d equal to 10 nM) , and an additional 3-fold by round 12 (K_d equal to 4 nM) .

Forty different individuals were isolated after 12 cycles (Fig. 18) (SEQ ID NOS:150-186) . Of the 40 different individuals isolated after 12 cycles, 3 of every 4 contained the pentamer CCCCT (or a variation of this pentamer) in the central 35 nucleotide cassette (Fig. 18) . The sequence of the invariant 3' end of each molecule in the library was AGGGG, and when paired to the internal CCCCT, the resulting duplex mimicked a primer:template junction substrate recognized naturally by the enzyme. A more careful analysis revealed additional base pairing: 13 of the 40 paired at least 6 nucleotides, 9 paired at least 7, 3 paired at least 8, and 1 contained the sequence CCCCTGTAG pairing with the 3' terminal CTGCAGGGG at 9 positions. If we assembled a collection of 40 randomly-chosen individuals from the degenerate library, the expected distribution of individuals able to form a duplex with the 3' terminus would be: 20 pairing 5 nucleotides, 4 pairing 6, 2 pairing 7, 1 pairing 8, and 0 pairing 9 (see infra) . This overrepresentation of junctions in the degenerate library suggested additional components were required for high affinity binding.

Our decision to isolate and sequence individual members of the enriched library after 12 SELEX cycles was made based on the enrichment profile shown in Fig. 17, where the small affinity change seen between selections ten and twelve suggested sufficient enrichment had occurred. Upon examination of the sequences, two observations were made that led us to believe that further rounds were necessary. First, of the 37 individuals isolated from the enriched library, only three were represented more than once, and none was represented more than two times. Sequence redundancy is often an indicator of sufficient enrichment, as highly represented sequences are believed to possess a component conferring a selectable advantage, ultimately resulting in their enrichment to a significant fraction of the selected library. Second, the majority of molecules selected by HIV-1 RT had the potential to form structures mimicking primer:template junctions. At first, the preference for junctions was discouraging as we hoped to identify ligands with complex, interesting secondary structures, but an analysis of sequence representation (discussed infra) suggested that complex ligands did exist in the degenerate library and might be found with a few more selection cycles.

While most of the selected ligands had the potential to form a primer:template junction, the sequences forming the duplex varied widely among individuals, most often forming imperfect helices with no apparent similarities. Because the frequency of individuals in the degenerate library with this characteristic was very high, specific binding to HIV-1 RT had to depend on more than the presence of a junction. If we accept G:T annealing as a stable base- pair, one of every 2 molecules in the degenerate library possessed a 5 base-pair junction. (This number was derived by calculating the fraction of pentamers with the sequence C/T-C/T-C/T-C/T-T, 1 in 64, multiplying by 31 to account for the number of windows a 35N region provides for a pentamer.) Similar calculations reveal that 1 in 630 individuals in the degenerate library could form a 10 base-pair junction, 1 in 4 X 10⁵ a 15 base-pair junction, and 1 in 2.7 X 10⁸ a 20 base-pair junction. The distribution of junction lengths of the round 12 library was unimpressive knowing that approximately 70% of the degenerate library consisted of ligands containing perfect junctions 5 base-pairs long or greater. Clearly, more complex ligands with higher affinity existed in the degenerate library, but were severely outnumbered by the remarkably high representation of individuals (with reasonable affinity for HIV-1 RT) containing a primer:template junction. The tremendous competition for available target binding sites increased the number of cycles necessary to enrich the higher affinity individuals to a sufficient fraction of the population, as predicted by Irvine, D.C., Tuerk, C, and Gold, L. (1991) J. Mol. Bio. 222:739-76. We performed three more cycles to enrich for molecules possessing binding features in addition to (or instead of) the stable primer:template junction. Individuals isolated from this round 15 library are herein referred to as RT "N, " where "N" represents the ligand number corresponding to the sequences shown in Figure 19 (SEQ ID NOS:187-216) . Only after three additional SELEX cycles did the underrepresented, but structurally more complex individuals surpass those lower affinity members possessing junctions. The majority of individuals in the round 15 library were unable to form primer:template junctions with the 3' terminus, but did have the potential to form ordered structures, primarily long helices with specific interruptions. Individuals able to form junctions that survived the three extra cycles each had additional components that increased their affinity relative to the round 12 library. Of the 30 different individuals isolated from the final population, only 1 of every 3 mimicked a primer:template junction, and those that did shared additional regions of similarity; for example, one subset had in common the octamer GCGTGCTG immediately upstream, and the nonomer AAAGGTGAT immediately downstream of the CCCCT pentamer (Fig. 19) . Replacement of the conserved upstream octamer with (dA) 8 resulted in a ligand with an affinity for HIV-1 RT as poor as the degenerate library (data not shown) . Compared with the isolates of the round 12 library, more members of the round 15 library were multiply represented (RT6 (SEQ ID NO:201) was represented 7 times, RT8 (SEQ ID NO:204) 4 times, RT12 (SEQ ID NO:192) 3 times, etc.) , indicating a more highly enriched representation of HIV-1 RT binders existed after 15 cycles . The high number of redundant sequences and conserved elements in the round 15 library indicated that further enrichment was unnecessary. The three additional cycles resulted in a decrease in the apparent K_d of the library to 2 nM, a total increase in affinity of 700-fold over the degenerate library. The isolates from this library were classified into subsets with common sequence elements. At least one from each subset (for a total of 8) was chosen for further characterization.

HIV-1 RT Binders Characterized by Long Interrupted Helices. The primary sequence diversity between subsets suggested that if there was a common element responsible for the affinity, it existed at a higher level of structure. Unfortunately, a reliable set of rules characterizing the folding of ssDNA molecules has not been elucidated, restricting us to use of the best tool available, an algorithm that uses rules for RNA folding to predict secondary structure (Jaeger, J. A. et al . (1989) Proc. Natl. Acad. Sci., U.S.A. 86:7706- 7710; Zuker, M. (1989) Science 24_4:48-52) . Potential structures offered by this algorithm for each of the eight ssDNA ligands are illustrated in Figs. 20A-20H. Optimal and suboptimal structures were compared within each group, and conserved structural elements were used to predict functional binding motifs. All of the ligands have the potential to form structures characterized by a high degree of base pairing, often making extensive use of the invariant regions to form long helices interrupted by mismatches, bulges, and internal loops. Ligands RT10 (SEQ ID NO:200) , RT12 (SEQ ID NO:192) , and RT26 (SEQ ID N0:188) are able to pair their 3' terminal AGGGG with an internal CCCCT to form an intramolecular primer:template junction. Of particular interest is the helix of RT26 (SEQ ID NO:188) , containing a potential internal loop with an AA opposite a CG as shown in Figure 21A. This motif can also be formed in ligand RTl (SEQ ID NO:215) , as well as variants in ligands RT4 (SEQ ID NO:208) (an AA opposite an AG) , RT8 (SEQ ID NO:204) (a CAA opposite a TAG) , and RT36 (SEQ ID NO:211) (an AA opposite an A) (Figs. 21B-21E) .

Binding Curves Confirm High Affinity of Individual Ligands. Affinity values of each of the eight chosen isolates for HIV-1 RT were measured using the filter binding assay described in Example 20 (Figs. 22A-22C) . The RNA pseudoknot inhibitor reported in Tuerk, C. et al . (1992) Proc. Natl. Acad. Sci., U.S.A. 89:6988-6992 and U. S. Patent Application No. 07/964,624 (see Fig. 23) had an affinity of 5 nM under our conditions, while that of the degenerate library (RO) is 1400 nM. Isolates RTl (SEQ ID NO:215) and RT26 (SEQ ID NO:188) exhibited the highest affinity having a K_d value of approximately 1 nM, while the others ranged from 2 to 11 nM. Differences in maximum percent bound likely reflect competing ligand structures with lower affinity. No correlation exists between representation in the fully-enriched library (see Fig. 19) and affinity for HIV-1 RT, as ligand RTl (SEQ ID NO:215) (K_d = 1 nM) , represented once, has a higher affinity than RT6 (SEQ ID NO:201) (K_d = 5 nM) , represented 7 times. Indicated dissociation constants were determined as in Example 20. No significant correlation was observed between the affinity of a molecule and the subset into which it was classified in Figure 19, as the three highest affinity ligands (RTl (SEQ ID NO:215), RT12 (SEQ ID NO:192) , and RT26 (SEQ ID NO:188) were each classified into different subsets. However, both RTl (SEQ ID NO:215) and RT26 (SEQ ID NO:188) contain the internal loop structure shown in Figures 21A and B, suggesting a possible participation of this motif in conferring high affinity upon ligands that possess it. Intramolecular Extension Verifies Secondary

Structure Predictions. The isolates possessing an intramolecular primer:template junction (RT10 (SEQ ID NO:200) , RT12 (SEQ ID NO-192), and RT26 (SEQ ID NO:188)) were assayed for the ability to be extended from their 3' termini by a variety of polymerases. The results for RT26 are shown in Figure 24. When extended with a saturating concentration of HIV-1 RT, initiation was nearly 100%, while extension proceeded only 5-8 nucleotides before premature termination occurred. AMV-RT initiated only 50%, but extension proceeded to the end of the template. With Sequenase T7 DNA polymerase, both initiation and extension went to completion. The sequence pattern created by extending with Sequenase in the presence of ddATP confirmed the proposed annealing site of the 3' end of RT26 (SEQ ID NO:188) . This was also true for RT10 (SEQ ID NO:200) and RT12 (SEQ ID NO:192) (data not shown) .

The premature terminations seen when extending RT26 with HIV-1 RT appear to be specific for that enzyme. Both products (premature and complete) were isolated and found to have 100-fold lower affinity for HIV-1 RT than the unextended RT26 (SEQ ID NO:188) (data not shown) . HIV-1 RT is less processive than AMV RT and Sequenase, and this lack of processivity might explain the premature termination, although using a saturating concentration of enzyme should have reduced this effect. Two alternative explanations for the premature termination are that addition of the 5-8 templated nucleotides to the 3' end of RT26 (SEQ ID NO:188) creates a low-affinity product, resulting in the release of enzyme more frequently than addition of the next nucleotide, or a trapped product unable to release enzyme or be further extended. Premature termination of RT26 (SEQ ID NO:188) extension occurred within the stem of a potential hairpin, suggesting termination was simply a result of interference by secondary structure; however, similar premature terminations occurred with RT10 (SEQ ID NO:200) and RT12 (SEQ ID NO:192) (data not shown) , neither of which occurred at positions stabilized by secondary structure. A groove on the surface of HIV-1 RT, shown by the

X-ray structure to extend from the polymerase catalytic site to the RNase H active site (Kohlstaedt, L. A. et al . (1992) Science 256 :1783-1790; Jacobo-Molina, A. et al . (1993) Proc. Natl. Acad. Sci., U.S.A. 90:6320-6324; Arnold, E. et al . (1992) Science 357:85-89; Krug, M. S. and Berger, S. L. (1991) Biochemistry £0=10614-10623) , is the best candidate for the protein region contacted by the selected DNA ligands. The ability of RT10 (SEQ ID NO:200) and RT12 (SEQ ID NO:192), and RT26 (SEQ ID NO:188) to be extended demands that the 3' end of these ligands be present in the polymerase active site when they are bound, likely positioned there by interactions between the helix and the protein groove.

Inhibition of Polymerase Activity Suggests Interaction at Substrate Binding Site and/or Active

Site. The ability of each of the 8 isolates to inhibit the RNA-dependent DNA polymerase activity of HIV-1 RT was assayed by measuring the decrease in extension product formation from a primer:template substrate as a function of inhibitor concentration (Figs. 25A-25I) . The substrate for the inhibition assay was a DNA:RNA heteroduplex consisting of an 18 nucleotide end-labeled DNA primer identical in sequence to the 3' end of tRNA^lys- ³ annealed to a 30 nucleotide RNA template whose sequence matches the genomic primer binding site and the first twelve transcribed nucleotides. Extension reactions were performed as described in Example 20 in the presence of 0, 81, 27, 9, 3, and 1 nM inhibitor as indicated in Figure 25.

The two bands on the gels are the unextended DNA primer migrating as an 18-mer, and the extended DNA product migrating as a 30-mer. The percent of primer extended as a function of inhibitor concentration is plotted for each inhibitor. K_± values were determined using a least-squares algorithm to fit the data points to Equations 4 and 5 of Example 20. We report these Ki' values rather than true K_L ' values because they were not determined with a standard Michaelis-Menten kinetic assay (comparing double-reciprocal plots of reaction velocity as a function of substrate concentration in the presence and absence of inhibitor) . However, the correlation between the Ki' and K_d values suggests that the mechanism of inhibition may be a competition between the inhibitory ligand and the substrate for the nucleic acid binding site and/or polymerase active site of RT, although this has not been tested directly.

Almost no inhibition was seen with as high as 81 nM of the degenerate ssDNA library present (R0, >_. 3 μM) . The RNA pseudoknot (RNA pk) inhibited the activity of HIV-1 RT with a K^ value of 4.7 nM under our conditions, consistent with the K_d value shown in

Figure 22 and Tuerk, C. et al . (1992) Proc. Natl. Acad. Sci., U.S.A. £9:6988-6992. The Ki' values of the seven ssDNA ligands assayed (only RTl (SEQ ID NO:215) and RT26 (SEQ ID NO:188) are shown) were also consistent with the K_d values shown in Figures 22A-22C. Clones RTl (SEQ ID NO:215) and RT26 (SEQ ID NO:188) were the most potent inhibitors of the RNA-dependent DNA polymerase activity of HIV-1 RT, having K_±' values of 0.3 nM and 2.7 nM, respectively. The Ki' values of RT4 (SEQ ID NO:208) , RT6 (SEQ ID NO:201) , RT8 (SEQ ID

NO:204) , RT10 (SEQ ID NO:200), and RT36 (SEQ ID NO:211) are 4.1 nM, 30 nM, 13 nM, 62 nM, and 6.5 nM, respectively. The K_± ' value of RT12 (SEQ ID NO:192) was not calculated. The correlation between the Ki' and K_d values suggests that the mechanism of inhibition may be a competition between the inhibitory ligand and the substrate for the nucleic acid binding site and/or polymerase active site of RT.

EXAMPLE 22. SUICIDE INHIBITORS OF HIV-1 RT

The specificity and high affinity for HIV-1 RT exhibited by these ssDNA ligands make them good candidates for suicide inhibitors of HIV-1 RT. This is accomplished by synthesizing a particular ssDNA ligand to HIV-1 RT, incorporating at specific positions nucleotide analogs possessing a reactive group able to covalently crosslink the ligand to HIV-1 RT upon binding. This attachment event would render the enzyme permanently non-functional. Reactive groups are chosen to utilize the specificity of the ligands for HIV-1 RT, being reactive only with HIV-1 RT and only when in close proximity (i.e., only when bound) . We have shown that HIV-1 can catalyze addition of a nucleotide to the 3' end of RT10 (SEQ ID NO:200) , RT12 (SEQ ID NO:192) , and RT26 (SEQ ID NO:188) . The ability of the existing HIV-1 RT ligand to extend by addition of a nucleotide to the 3'-end can be exploited for mechanism-based suicide inhibition of the enzyme. This will result in covalent linking of the ligand to the target.

The crucial step in addition of a nucleotide onto the 3'-end of the existing ligand is the abstraction of the proton from the 3'-hydroxyl group by a base associated with the enzyme. Proton extraction or activation of the 3'-hydroxyl aids in the attack of the αt-phosphorous of the incoming nucleoside triphosphate. A 3'-terminal nucleoside analog can be designed, that exploits base-activation of the 3'-hydroxyl group to form a reactive intermediate. This species, which is generated in close proximity to the enzyme surface, is then ready to accept an enzyme nucleophile to generate a covalent link.

oligo

The terminal 3' -nucleotide is modified to bear a leaving group at the 2'-position in anti stereoconfiguration to the 3'-hydroxyl. A typical leaving group could be a halogen, an acetyl group, a sulfonate group, a carbonate group, an acetamide group or any other leaving group. Upon deprotonation of the 3'-hydroxyl by the enzyme a 2' ,3' -epoxide is formed on the α-face of the nucleoside. This epoxide is labile enough to be attacked from the β-face of the furanose by any adjacent nucleophile on the enzyme. This process results in a covalent link between the enzyme and the ligand.

To increase specificity of inhibition, covalent crosslinking could be coupled to activity of the enzyme. If RT10 (SEQ ID NO:200), RT12 (SEQ ID NO:192), and RT26 (SEQ ID NO:188) were synthesized with a nucleotide analog at its 3' end containing an electron withdrawing group at the 2' carbon, catalytic addition of a nucleotide triphosphate (step 1 of Fig. 30) would result in a spontaneous elimination event, releasing the newly added nucleotide and yielding an electrophilic carbon at the 3' position of the sugar polarized by the electron withdrawing group at the 2' position (step 2 of Fig. 30) . The reactive 3' carbon would be available for attack by any good nucleophilic group in the vicinity, resulting in the formation of a covalent crosslink between the protein and the ligand (step 3 of Fig. 30) . Because this reaction is dependent on catalysis by HIV-1 RT, these inhibitors would specifically target active enzyme. It is possible that RT10 (SEQ ID NO:200) , RT12 (SEQ ID N0.192) , and RT26 (SEQ ID NO:188) interact with HIV-1 RT in such a way that there are two aspartic acid residues and one tyrosine near enough to perform the reaction.

EXAMPLE 23. ESSENTIAL ELEMENTS OF RTl

Biased Synthesis SELEX Identifies Essential Elements of RTl. From a library of RTl mutants, synthesized as described in Example 20, we selected those maintaining a high affinity for HIV-1 RT. In six SELEX cycles the affinity of the library increased almost 1000-fold, from 1500 nM to approximately 2 nM (data not shown) . About one half of the 32 isolates had a primary sequence consistent with the predicted secondary structure of RTl (SEQ ID NO:215), while the other half adopted alternative structures with equally high affinity. The sequences of the isolates similar in structure to RTl are shown in Figure 26 (SEQ ID

NOS:220-235) . The acceptability of mutations varied with position: mutations in the 3' region of the randomized cassette (positions 29-35) were most tolerated, while those in the 5' region (positions 1-7) eliminated ability to bind HIV-1 RT and were selected against. Conservation of positions 1-3 and 6-9 support the internal loop duplex structure comprising the predicted 5' domain of RTl when paired with the 5' invariant primer-binding region. Additional support for the base-pairing pattern in this domain is provided by the acceptability of the A to G substitution at position 2, which is able to maintain the base pair with the invariant T. The small hairpin predicted to exist in the central domain of RTl (SEQ ID NO:215) is not supported by the results of this experiment. In the stem of the proposed hairpin, many substitutions disrupting the base-pairing pattern were acceptable, and alternative structures were preferred. No predicted structure could accommodate each of the selected individuals, suggesting the absence of secondary structure in this region. HIV-1 RT might recognize specific unpaired residues of this central domain of RTl, possibly those indicated in the consensus illustrated in Figure 26B (SEQ ID NO:236) .

Only 49 Nucleotides Required of RTl for High Affinity. Truncated versions of ligand RTl (SEQ ID NO:215) were synthesized and predicted secondary structures are shown in Figures 27A-27C. The predicted secondary structures, using the RNA folding algorithm of Zucker, M. (1989) Science 244 :48-52 and Jaeger, J. A. et al . (1989) Proc. Nat. Acad. Sci. USA 86:7706- 7710, were refined by the results of the biased randomization experiment, in particular, the lack of secondary structure in the central region. These truncates were tested for their ability to bind HIV-1 RT with high affinity. RTlt30 (SEQ ID NO:238), composed of the first 30 nucleotides of RTl containing the internal loop duplex, showed no significant binding below 1 μM HIV-1 RT. However, addition of the next 19 nucleotides, comprising the central stem and loop motif, produced a 49-mer (RTlt49) (SEQ ID NO:237) which bound HIV-1 RT with an affinity of 4 nM, nearly as high as the full-length 81 nucleotide RTl. The relative affinities of RTl (SEQ ID N0.215), RTlt30 (SEQ ID NO:238) , and RTlt49 (SEQ ID NO:237) suggest that all of the specific binding components of RTl (SEQ ID NO:215) exist in the first 49 nucleotides and that while the internal loop motif is insufficient alone, it likely participates in the interaction with HIV-1 RT in combination with other specific binding components.

49-mer Inhibits HIV-1 RT Specifically. The inhibition assay described in Example 20 was also used to determine the specificity of inhibition of the RNA- dependent DNA polymerase activity of HIV-1 RT. Using ligand RTlt49 (SEQ ID NO:237) as the competitor, we compared in parallel the ability to inhibit the polymerase activity performed by HIV-1 RT, AMV RT, and MMLV RT. As illustrated in Figure 28, inhibition of primer extension was seen when performed with HIV-1 RT, but was not detectable when performed with AMV RT and MMLV RT, even at inhibitor concentrations as high as 81 nM. The lack of inhibition of AMV RT and MMLV RT possibly suggests that RTlt49 (SEQ ID NO:237) may have a lower affinity for these enzymes, requiring higher concentrations of RTlt49 to see an inhibitory effect on primer extension.

EXAMPLE 24. RTl COMPETES WITH RNA PSEUDOKNOT FOR RT Binding. The specific inhibition characteristics exhibited by both an RNA pseudoknot and a ssDNA ligand posed the question of whether two apparently dissimilar molecules, at least at the level of secondary structure, interact with HIV-1 RT at a common nucleic acid binding site. To test this, we measured the ability of the ssDNA ligand RTl (SEQ ID NO:215) to maintain its specific binding contacts with HIV-1 RT in the presence of high concentrations of RNA pseudoknot (see Fig. 23) . Figure 29 shows the competitive binding of RTl 215 (SEQ ID NO:215) and the RNA pseudoknot (RNA pk) (SEQ ID NO:217) . The percent of RTl bound in the presence of competitor relative the percent bound in the absence of competitor is plotted as a function of RNA pk concentration. The K_c value for RNA pk (3 nM) was determined using an algorithm that fit the data points to Equations 4 and 5 in Example 20, and was consistent with the Kd value (5 nM) measured using the nitrocellulose filter binding assay described in Example 20. As shown graphically in Figure 29, when a stoichiometric equivalent of RNA pk was added, approximately one half of the complexed RTl was displaced, and nearly all was displaced when a large excess of RNA was added. These results leave little doubt that binding of RTl (SEQ ID NO:215) and RNA pk (SEQ ID NO:217) to HIV-1 RT are mutually exclusive. However, with this assay we are unable to distinguish between an interaction of both ligands at a common site or an interaction of each at different sites, with a conformational change upon binding the first that prevents subsequent binding of the second.

EXAMPLE 25. A PHOSPHOROTHIOATE CAP RESULTS IN

STABILITY AGAINST 3 ' TO 5' EXONUCLEASES WITHOUT AFFECTING BINDING AFFINITY

A ssDNA ligand RTlt49PS (SEQ ID NO:239) consisting of ligand RTlt49 (SEQ ID NO:237) with a phosphorothioate cap added to its 3' end was synthesized by standard phosphoramidite chemistry by Operon (Alameda, California) . The cap is four thymine residues linked by a phosphorothioate backbone instead of the standard phosphodiester backbone. This ligand has the following sequence, where each residue designated by a small case letter is attached to its downstream neighbor by a phosphorothioate linkage:

5' -ATCCGCCTGATTAGCGATACTCAGAAGGATAAACTGTCCAGAACT TGGatttT-3'

The affinity of RTlt49PS for HIV-1 RT was compared with that of RTlt49 using a standard filter binding assay. The Kds were both found to be 5.5 nM, showing that the phosphorothioate cap added stability against 3' to 5' exonucleases without affecting binding affinity of the ligand for HIV-1 RT.

TABLE 1 Summary of SELEX experiment A selection cycle results.

Experiment A

Selection Counterselection Percent eluted cycle pmoles RNA ^a column ? ^b with DTT ^c

l^d 500 N 0.47

1 500 N 0.35

2 200 N 1.92

3 50 N 0.77

4 50 N 0.61

5 50 Y 2.57

6 50 Y 1.52

7 50 Y 2.27

8 50 Y 13.8

9 50 Y 19.8

10 50 Y 17.5

11 50 Y 19.4

12 50 Y 12.0

(a) pmoles of RNA applied to a 100-μl column volume of SP-Sepharose (80 μM SP) . (b) Whether a thiopropyl-Sepharose column counterselection step was incorporated into the selection scheme prior to affinity selection on SP-Sepharose is indicated by either N (no) or Y (yes) . (c) Percentage of the applied RNA that was eluted with ten column volumes of SP binding buffer containing 100 mM DTT following an extensive wash with 20 column volumes of SP binding buffer. (d) Results from a control experiment in which the initial random sequence RNA pool was applied to a thiopropyl-Sepharose column in the absence of substance P. TABLE 2 Summary of SELEX experiment B selection cycle results.

Experiment B

Selection Counterselection Percent eluted cycle ^a [SP] column ? ^c with DTT ^d

Ligand A13^e 80 mM N 9.97

1 80 mM N 1.01

2 80 mM N 0.92

3 20 mM N 1.87

4 20 mM N 5.52

5 20 mM N 3.85

6 20 mM N 4.46

7 20 mM N 29.2

8 5.0 mM Y 16.3

9 2.5 mM Y 10.5

10 1.3 mM Y 8.00

11 1.3 mM Y 12.6

12 1.3 mM Y 14.4

(a) PCR mutagenesis of the ligand pool (of ligand A13 (SEQ ID NO:22) for selection cycle 1) preceded selection cycles 1-6; standard PCR amplification of the ligand pool preceded selection cycles 7-12. (b) Concentration of substance P contained on the SP- Sepharose affinity matrix as a function of column volume (100 μl) . Binding reactions occurred in well- mixed 1 ml suspensions. (c) Whether a thiopropyl- Sepharose column counterselection step was incorporated into the selection scheme prior to affinity selection on SP-Sepharose is indicated by either N (no) or Y (yes) . (d) Percentage of the applied RNA that was eluted with 10 column volumes of SP binding buffer containing 100 mM DTT following an extensive wash with greater than 20 column volumes of SP binding buffer. (e) Results from a control experiment with non-mutagenized ligand A13. TABLE 3 The starting ss DNA template and the 3' and 5' PCR primers used in SELEX experiments to select RNA ligands to HIV-1 integrase. ssDNA Template

5' -GCCGGATCCGGGCCTCATGTCGA [3ON]TTGAGCGTTTATTCTGAGCTCCC-

3'

(SEQ ID NO:140)

3'PCR Primer T7 Promoter

5' -CCGAAGCTTAATACGACTCACTATAGGGAGCTCAGAATAAACGCTCAA-

Hindlll (SEQ ID NO:141)

PCR Primer

5' -GCCGGATCCGGGCCTCATGTCGAA-3'

Bam HI (SEQ ID NO:142)

TABLE 4 Selected sequences to HIV-1 integrase isolated from the library after 18 rounds

SELEX.

Group I SEQ ID NO:

P5 (18) GGGAGCUCAGAAUAAACGCUCAACCAGUCUUGUGGCUUUGAAAGAGAGGAGUGUUCGACAUGAGGCCCGGAUCCGGC 84

P54 (5) GGGAGCUCAGAAUAAACGCUCAACCAGUCUUGUGGCAUUGAAAGAUAGGUGUGUUCGACAUGAGGCCCGGAUCCGGC 85

P23 (5) GGGAGCUCAGAAUAAACGCUCAACCAGUCUUAUGGCGUUGCAAGAUAGGGGCGUUCGACAUGAGGCCCGGAUCCGGC 86

PI (3) GGGAGCUCAGAAUAAACGCUCAACCAGUAUUAUGGCUUUGAGAGAGAGGUGCGUUCGACAUGAGGCCCGGAUCCGGC 87

A15 (3) GGGAGCUCAGAAUAAACGCUCAACCAGUCUUGUGGCUUUGU.AAGAGAGGAGUGUUCGACAUGAGGCCCGGAUCCGGC 88

P29 (2) GGGAGCUCAGAAUAAACGCUCAACCAGUCUUAUGGCUUUGAAAGUGAGGAGUGUUCGACAUGAGGCCCGGAUCCGGC 89

Group II

A54 (8) GGGAGCUCAGAAUAAACGCUCAACGGCACAGGGGUUGUAUCCUCCGGGACGAAUUCGACAUGAGGCCCGGAUCCGGC 90

P47 (5) GGGAGCUCAGAAUAAACGCUCAACGGCACAGGGCCUGUAUCCUCCGGGCCGAAUUCGACAUGAGGCCCGGAUCCGGC 91

P56 (4) GGGAGCUCAGAAUAAACGCUCAACGGCAUAGGGGUUGUAUCCUCCGGGACGAAUUCGACAUGAGGCCCGGAUCCGGC 92

P64 (2) GGGAGCUCAGAAUAAACGCUCAACGGCACCGGGGCUGUAUCCUCCGGCACGAAUUCGACAUGAGGCCCGGAUCCGGC 93

All (1) GGGAGCUCAGAAUAAACGCUCAAAGAUUGAAUGGGGGUAACCAACGGGAGAUUCGACAUGAGGCCCGGAUCCGGC 94

Group III ^

Al GGGAGCUCAGAAUAAACGCUCAAGUCAAUCAUCGAUGUCCUGUGCCCUAGGGCUUCGACAUGAGGCCCGGAUCCGGC 95

A7 GGGAGCUCAGAAUAAACGCUCAAGUCAAUCUUCGAUGUGCUGUGCCCGAUGAAUUCGACAUGAGGCCCGGAUCCGGC 96

P57 GGGAGCUCAGAAUAAACGCUCAAGUCAAUCAUCGAUGUGCUGUGCCCGAUAAAUUCGACAUGAGGCCCGGAUCCGGC 97

A29 GGGAGCUCAGAAUAAACGCUCAAGUCAAUUAUCGAUGUGCUGUGCCCGAUCAAUUCGACAUGAGGCCCGGAUCCGGC 98

P60 GGGAGCUCAGAAUAAACGCUCAAGUCAAUUAUCGAUGUGCUGUGCCCGAUGAAUUCGACAUGAGGCCCGGAUCCGGC 99

A25 GGGAGCUCAGAAUAAACGCUCAAGUCAAUUAUCAAAGUGCGGAACCCUAUGAAUUCGACAUGAGGCCCGGAUCCGGC 100

PI9 GGGAGCUCAGAAUAAACGCUCAAGUCGAGGCCCGGAUGUGCUGUGCCCUGGGAUUCGACAUGAGGCCCGGAUCCGGC 101

Al8 GGGAGCUCAGAAUAAACGCUCAAGUCCUAAUCCCUAAUGUGAUCUGAUGAAUUCGACAUGAGGCCCGGAUCCGGC 102

A46 GGGAGCUCAGAAUAAACGCUCAAGCCCCCGGUUGAAGACUUGUAAUGCCCUAAUUCGACAUGAGGCCCGGAUCCGGC 103

P55 GGGAGCUCAGAAUAAACGCUCAAGUCUCGCAUUUAGACAGACCUGUGCCCUAAAUUCGACAUGAGGCCCGGAUCCGGC 104

A10 GGGAGCUCAGAAUAAACGCUCAAUGUUGAGUAAGACGAGCUGUGCCCUUAUUCGACAUGAGGCCCGGAUCCGGC 105

A23 GGGAGCUCAGAAUAAACGCUCAAUGGUUGUGAAAGAUGAGGUGAGCUCUUAUUCGACAUGAGGCCCGGAUCCGGC 106

TABLE 4 (CONTINUED)

Group III (CONTINUED) SEQ ID NO:

P20 GGGAGCUCAGAAUAAACGCUCAACGCACGACUAAGGAUGUGCUGUGCCCUUUAUUCGACAUGAGGCCCGGAUCCGGC 107

P38 GGGAGCUCAGAAUAAACGCUCAAUGUACGACUAAGCAUGUGCUGUGCCCUUUAUUCGACAUGAGGCCCGGAUCCGGC 108

A28 GGGAGCUCAGAAUAAACGCUCAAUGGACACUAGAUGAGGUGCGCUGUGCACAUUUCGACAUGAGGCCCGGAUCCGGC 109

P59 GGGAGCUCAGAAUAAACGCUCAAUUGGAACUCGAAAUGAUCUGCUGUGCCCAUUUCGACAUGAGGCCCGGAUCCGGC 110

P21 GGGAGCUCAGAAUAAACGCUCAAUGUUUGGAGAAGAGCCGUGCCCUCUAGACAUUCGACAUGAGGCCCGGAUCCGGC 111

P27 GGGAGCUCAGAAUAAACGCUCAAUGUUUGGAGGAGAGCCGUGCCCUCUAGACAUUCGACAUGAGGCCCGGAUCCGGC 112

P22 GGGAGCUCAGAAUAAACGCUCAAGUUUGGAGGAGUGAUGUCCUCUCUAGGCAUUCGACAUGAGGCCCGGAUCCGGC 113

A14 GGGAGCUCAGAAUAAACGCUCAAGAAGUGCUGUGCCCUUGACCGUUUUAUUUCUUCGACAUGAGGCCCGGAUCCGGC 114

A39 GGGAGCUCAGAAUAAACGCUCAAGAUGUGCUGUGCCCUUGAGUCGUUUCCAGUUCGACAUGAGGCCCGGAUCCGGC 115

A64 GGGAGCUCAGAAUAAACGCUCAAGAUGUGCUGUGCCCUUCCUCCGUUUCCAAUUCGACAUGAGGCCCGGAUCCGGC 116

P37 GGGAGCUCAGAAUAAACGCUCAAGAUGUGCUGUGCCCUUGGCCAGUUUCCAAUUCGACAUGAGGCCCGGAUCCGGC 117

P25 GGGAGCUCAGAAUAAACGCUCAAUCGGUAUGUGCUGUGCCCCCGAGAGUUCGUUCGACAUGAGGCCCGGAUCCGGC 118

P39 GGGAGCUCAGAAUAAACGCUCAAGCGGAUGUGCGGUGCCCUGCUUAAACGUUGUUCGACAUGAGGCCCGGAUCCGGC 119

A2 GGGAGCUCAGAAUAAACGCUCAAGCGCUGCCUCAGGUAAUGCCCUUAGAAAGUUCGACAUGAGGCCCGGAUCCGGC 120 ,

A20 GGGAGCUCAGAAUAAACGCUCAAGCGAUCGACUGCAUCAUAUGGCACGAGAUUCGACAUGAGGCCCGGAUCCGGC 121

A48 GGGAGCUCAGAAUAAACGCUCAAGUGGUGAAUCAGUGCGUGUGUGGCCUAGAUUCGACAUGAGGCCCGGAUCCGGC 122

A16 GGGAGCUCAGAAUAAACGCUCAAUGUCCGAAAAUCACGUUGCUGCAGACACAUUCGACAUGAGGCCCGGAUCCGGC 123

Al7 GGGAGCUCAGAAUAAACGCUCAAACAUCGAUGACCGGAAUGCCGCACACAGAGUUCGACAUGAGGCCCGGAUCCGGC 124

A45 GGGAGCUCAGAAUAAACGCUCAAUAAGCCUCACGUUUGUCUGAACAGGAUCGUUCGACAUGAGGCCCGGAUCCGGC 125

A47 GGGAGCUCAGAAUAAACGCUCAAGCCUCACUGUUGUAUUGUGCCGCAUGGCAUUCGACAUGAGGCCCGGAUCCGGC 126

P42 GGGAGCUCAGAAUAAACGCUCAAUCCAUGUUCGAUAUACAGGAUGGAAAGGUUCGACAUGAGGCCCGGAUCCGGC 127

A5 GGGAGCUCAGAAUAAACGCUCAAUGUCCUUAACUUGCUACUUCACGCUGUACUUCGACAUGAGGCCCGGAUCCGGC 128

A55 GGGAGCUCAGAAUAAACGCUCAAUGUCCGUUUUAUGUCAAAUGUAUUUCGUAAUUCGACAUGAGGCCCGGAUCCGGC 129

A9 GGGAGCUCAGAAUAAACGCUCAAGAUCCGCAGUAACUGAUAAUGUUAAAGUACUUCGACAUGAGGCCCGGAUCCGGC 130

A42 GGGAGCUCAGAAUAAACGCUCAAUAGCCGGGUCAAGAAAGCCGGACAGUGUUAUUCGACAUGAGGCCCGGAUCCGGC 131

A24 GGGAGCUCAGAAUAAACGCUCAAGAGGCUCAACCCUUACUGCAUGCUGGUCAAUUCGACAUGAGGCCCGGAUCCGGC 132

A53 GGGAGCUCAGAAUAAACGCUCAAGUUCACAAGAGGAAACCAUUAAUGCUAAUUCGACAUGAGGCCCGGAUCCGGC 133

TABLE 4 (CONTINUED)

Group III (CONTINUED) SEQ ID NO:

P32 GGGAGCUCAGAAUAAACGCUCAACGACGCUAAACGUAGCUUGGUUGUGUAUUCGACAUGAGGCCCGGAUCCGGC 134

P41 GGGAGCUCAGAAUAAACGCUCAAUGACUAUGGGCUAGACUGCUUGGUGAAUUCGACAUGAGGCCCGGAUCCGGC 135

A40 GGGAGCUCAGAAUAAACGCUCAAACCCCUGACGCGCACGUAUAGCUGACUAAUUCGACAUGAGGCCCGGAUCCGGC 136

PI2 GGGAGCUCAGAAUAAACGCUCAACCUGAGAACUGAAGCCCUCGUCUGCCGUAAUUCGACAUGAGGCCCGGAUCCGGC 137

A60 GGGAGCUCAGAAUAAACGCUCAAGUCGACACGUACUGAGGUCGCGGAAGUAUUCGACAUGAGGCCCGGAUCCGGC 138

Numbers in parentheses indicate frequency of clone. Sequences with an A followed by a number were isolated from the low salt experiment, and sequences with a P followed by a number were isolated from the high salt experiment. Bases in bold type are fixed sequences used for PCR.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: GOLD, LARRY

NIEU LANDT, DAN ECKER, MATTHEW SCHNEIDER, DANIEL J. FEIGON, JULI ALLEN, PATRICK SULLENGER, BRUCE A. DOUDNA, JENNIFER, A. (ii) TITLE OF THE INVENTION: HIGH-AFFINITY LIGANDS OF INSULIIN RECEPTOR ANTIBODIES, TACHYKININ SUBSTANCE P, HIV INTEGRASE AND HIV-1 REVERSE TRANSCRIPTASE • (iii) NUMBER OF SEQUENCES: 239 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Swanson & Bratschun, L.L.C.

(B) STREET: 8400 E. Prentice Avenue, Suite 200

(C) CITY: Englewood

(D) STATE: Colorado

(E) COUNTRY: USA

(F) ZIP: 80111

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 MG storage

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/238,863

(B) FILING DATE: 06-MAY-1994 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/248,632

(B) FILING DATE: 24-MAY-1994

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/303,362

(B) FILING DATE: 09-SEPTEMBER-1994 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/361,795

(B) FILING DATE: 21-DECEMBER-1994 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/117,991

(B) FILING DATE: 08-SEPTEMBER-1993 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/931,473

(B) FILING DATE: 17-AUGUST-1992 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/964,624

(B) FILING DATE: 21-OCTOBER-1992 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/536,428 (B) FILING DATE: ll-JUNE-1990 (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/714,131

(B) FILING DATE: 10-JUNE-1991

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 07/536,428

(B) FILING DATE: ll-JUNE-1990 (viii)ATTORNEY/AGENT INFORMATION:

(A) NAME: Barry J. Swanson

(B) REGISTRATION NUMBER: 33,215

(C) REFERENCE/DOCKET NUMBER: NEX17/PCT (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (303) 793-3333

(B) TELEFAX: (303) 793-3433

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : GCGGAAGCGU GCUGGGCCNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNCA UAACCCAGAG GUCGAU 76

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 51 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : GGGGGAATTC TAATACGACT CACTATAGGG AGAGCGGAAG CGTGCTGGGC 50 C 51

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGGGGATCC ATCGACCTCT GGGTTATG 28

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GGGAGAGCGG AAGCGUGCUG GGCCGAUGUC GUAGAGCUAC AACUGAAGGC 50 AUAACCCAGA GGUCGAUGGA UC 72

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : GGGAGAGCGG AAGCGUGCUG GGCCGAUGUC AUAGAGCUAC AACUGAAGAC 50 AUAACCCAGA GGUCGAUGGA UC 72

(2) INFORMATION FOR SEQ ID NO: 6 :

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 : GGGAGAGCGG AAGCGUGCUG GGCCUCGCCG AGCUACAACU GAAGAGGUCA 50 ACGAGCACGC GAUACAUAAC CCAGAGGUCG AUGGAUC 87

(2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 71 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 : GGGAGAGCGG AAGCGUGCUG GGCCUUGUCA UAGAGCUACA ACUGAAGACA 50 UAACCCAGAG GUCGAUGGAU C 71

(2) INFORMATION FOR SEQ ID NO: 8 :

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 72 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GGGAGAGCGG AAGCGUGCUG GGCCAUGCCA ACAAAGCUAC AACUGAAGGC 50 AUAACCCAGA GGUCGAUGGA UC 72

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : GGGAGAGCGG AAGCGUGCUG GGCCUGUCAU AGAGCUACAA CUGAAGACAU 50 AACCCAGAGG UCGAUGGAUC 70

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GGGAGAGCGG AAGCGUGCUG GGCCUAUCAU AGAGCUACAA CUGAAGACAU 50 AACCCAGAGG UCGAUGGAUC 70

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGAGAGCGG AAGCGUGCUG GGCCUCGCCG AGCUACAACU GAAGAGGUCA 50 ACGAGCACGC GAUUCAUAAC CCAGAGGUCG AUGGAUC 87

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGGAGAGCGG AAGCGUGCUG GGCCCGUGGA GACAUGUAGA GCUUCAACUG 50 AAAUGUGUCA CGAGCAUAAC CCAGAGGUCG AUGGAUC 87

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 79 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GGGAGAGCGG AAGCGUGCUG GGCCUCGCAA AGCUACAACU GAAGAGGUGU 50 GUCGAACAUA ACCCAGAGGU CGAUGGAUC 79

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 82 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGGAGAGCGG AAGCGUGCUG GGCCUGUCAU AGAGCUACAA CUGAAGGGCC 50 GCUCCAUUUC AUAACCCAGA GGUCGAUGGA UC 82

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GGGAGAGCGG AAGCGUGCUG GGCCUCRYAR AGCUACAACU GAAGRCAUAA 50 CCCAGAGGUC GAUGGAUC 68

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CUGGGCCGAU GUCGUAGAGC UACAACUGAA GGCAUAACCC AG 42

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CGUGGAGACA UGUAGAGCUU CAACUGAAAU GUGUCACG 38

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 18 :

GCCGGATCCG GGCCTCATGT CGAANNNNNN NNNNNNNNNN N-S1NNNNNNNN 50

--TOIrø-SINNNNN -SINNNNNNNNN --MISINNNNNNN NNNNTTGAGC GTTTATTCTG 100

AGCTCCC 107

(2 ) INFORMATION FOR SEQ ID NO : 19 :

( i ) SEQUENCE CHARACTERIZATION :

(A) LENGTH : 48 base pairs

(B) TYPE : nucleic acid

( C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CCGAAGCTTA ATACGACTCA CTATAGGGAG CTCAGAATAA ACGCTCAA 48

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20 GCCGGATCCG GGCCTCATGT CGAA 24

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

GGGAGCUCAG AAUAAACGCU CAANNNNNNN NNNNNNNNNN NNNNNNNNNN 50

NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNUUCGACA UGAGGCCCGG 100

AUCCGGC 107 (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERIZATION: .

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22

GGGAGCUCAG AAUAAACGCU CAAGGGCAAC GCGGGCACCC CGACAGGUGC 50

AAAAACGCAC CGACGCCCGG CCGAAGAAGG GGAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:23

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Arg Pro Lys Pro Gin Gin Phe Phe Gly Leu Met

5 10

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24 Arg Pro Lys Pro Gin Gin Phe Phe Gly Leu Met Cys

5 10

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Cys Met Leu Gly Phe Phe Gin Gin Pro Lys Pro Arg

5 10

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 108 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

GGGAGCUCAG AAUAAACGCU CAAGAACAAG AUGGCAGUAA CGCAACCCAG 50

ACAGGAAAAA AACCCGACGC GCAAAAACAA CGGAUUCGAC AUGAGGCCCG 100

GAUCCGGC 108

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 109 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

GGGAGCUCAG AAUAAACGCU CAAGAACAAG AUGGCAGUGA CGCAACCCAG 50

ACAGGAAAAA AACCCGACGC GCAAAAAACA ACGGAUUCGA CAUGAGGCCC 100

GGAUCCGGC 109

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 111 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

GGGAGCUCAG AAUAAACGCU CAAGAACAAG AUGGCAAUAA CGCAACCCAG 50

ACAGGAAAAA AAAACCCGAC GCGCAAAAAA CAACGGAUUC GACAUGAGGC 100

CCGGAUCCGG C 111

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

GGGAGCUCAG AAUAAACGCU CAAGAAGCGA AAACAGAGGC GAGAGGAAAC 50

CUAAAACAGC GACGAAGCGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

GGGAGCUCAG AAUAAACGCU CAAGAAGCGA AAACAGAGGC GAGAGGAAAC 50

CUAAAACAGC GACGAAGUGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

GGGAGCUCAG AAUAAACGCU CAAGAAGCGA AGACAGAGGC GAGAGGAAAC 50

CUAAAACAGC GACGAAGUGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGGAGCUCAG AAUAAACGCU CAAGAAGUGA AAACAGAGGC GAGAGGAAAC 50 CUAAAACAGC GACGAAGCGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100 AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

GGGAGCUCAG AAUAAACGCU CAAGAAGAGA AAACAGAGGC GAGAGGAAAC 50

CUAAAACAGC GACGAAGCGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

GGGAGCUCAG AAUAAACGCU CAAGAACCGA AAACAGAGGC GAGAGGAAAC 50

CUAAAACAGC GACGAAGCGG CCACUGGUAU CUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

GGGAGCUCAG AAUAAACGCU CAACGCACGA CGCACCGUUA CAGGGGGGGA 50

AGAACCAACC CGAGCGCACG ACGGACCGAC GCUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

GGGAGCUCAG AAUAAACGCU CAACGCACGA CGCACCGUUA CAGGGGGGGA 50

AAAGCCAACC CGAGCGCACG ACGGACCGAC GCUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

GGGAGCUCAG AAUAAACGCU CAACGCACGA CGCACCGUUA CAGGGGGGGA 50

AAAAGCCAAC CCGAGCGCAC GACGGACCGA CGCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

GGGAGCUCAG AAUAAACGCU CAAAGGGCAA CGCGGGCACC CCGACAGGUG 50

CAAAACGCAC CGACGCCCGG CCGAAGAAGG GGAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

GGGAGCUCAG AAUAAACGCU CAAGCGAAAA GACGAAAAAA CCGACGACAC 50

UAGCGCGAUU CGGAAGACUA GCAACAACGA CACUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

GGGAGCUCAG AAUAAACGCU CAAAAGGAAG AAAACAGCAU AAUUAGGCAA 50

AAAGACAAAA ACAACAAAUA AAGAAAGAGC AUAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

GGGAGCUCAG AAUAAACGCU CAAACAAAAA ACAAACGAAA ACAUAAAAAU 50

AAAAUUAAAG UAGAAGCGCA AAGAUUAUUA CAAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 108 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GGGAGCUCAG AAUAAACGCU CAAAACUCAA UAUAAAGAAA ACGACAAAAC 50 AGAAUGAAGC CAAGAAAACA UACAAGAACG AAGCUUCGAC AUGAGGCCCG 100 GAUCCGGC 108

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

GGGAGCUCAG AAUAAACGCU CAACAAGCAA GGCAAUGCAA ACCCUUAGGU 50

CACAAGAACC GAUGAGGCUG UCCGGCACUU CAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

GGGAGCUCAG AAUAAACGCU CAACCAAACU AGGCUAUGGA AACCCUAAGG 50

CUAAUAAAGC CAAUGACGCC AUCCAGGUAC UUCUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

GGGAGCUCAG AAUAAACGCU CAAGAGACCU GGCAAUCGAA ACCCUAAGGA 50

UACAUAAUCC AAUGAGACCA UCCGGUCACU UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

GGGAGCUCAG AAUAAACGCU CAACGCUCCG GCAGUAGAAA CCCUAAGGUU 50

AUUAGACCAA UGAUGCCAUC CGGCCACAAC UUUCGACAUG AGGCCCGGAU 100

CCGGC 105

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGGAGCUCAG AAUAAACGCU CAACCUCCUG GCAGUAGAAA CCCUAAGGUC 50 AUUACGACCA AUGAUGCUAU CCAGGUACUU CAUUCGACAU GAGGCCCGGA 100 UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

GGGAGCUCAG AAUAAACGCU CAACGCUCCU GGCUGUGGAA ACCCUUAGGU 50

ACAAAAACCA AUGACGCCAU CUGGACAAUU CAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GGGAGCUCAG AAUAAACGCU CAACCAACCA GGCUAUGGAA ACCCUUAGGU 50

UAUAACAACC AAUGACGCCG UCCAGGUUCA UCUUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:

GGGAGCUCAG AAUAAACGCU CAACAGCAAG GCUAUAGAAA CCCUUAGGUC 50

AUAAAGACCA AUGAUGCCUU CCAGGUUCUU CUUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

GGGAGCUCAG AAUAAACGCU CAACGUACCG GCAAUCGAAA CCCUAAGGUU 50

ACAUAAACCA AUGAGGCCGC ACGGUCACUU CAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GGGAGCUCAG AAUAAACGCU CAAGAGACCA GGCUGAUGAA ACCCUUAGGC 50 UUAAUAACCA AUGAUGCCAU CCGGCAUACU UCAUUCGACA UGAGGCCCGG 100 AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 108 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

GGGAGCUCAG AAUAAACGCU CAACUUCCAA GGCAGUGGAA ACCCUAAGGU 50

CAAUAAUGAC CAAUGACGCC GCUCCGGUUC AACCUUCGAC AUGAGGCCCG 100

GAUCCGGC 108

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

GGGAGCUCAG AAUAAACGCU CAAGAGACCC GGCAAUAGAA ACCCUUAGGA 50

CACAAAGUCC AAUGAUGCCG UCCACAUACU UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 105 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

GGGAGCUCAG AAUAAACGCU CAACUCUGGG CUAUCGAAAC CCUUAGGAUA 50

CAAAAUCCAA UGAGGCCGAC CGGUAACAUU CUUCGACAUG AGGCCCGGAU 100

CCGGC 105

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:

GGGAGCUCAG AAUAAACGCU CAAAGUUCCU GGCAGUAGAA ACCCUAAGGU 50

CACUUAGACC AAUGAAGCCU UCCGGUUAUA UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 108 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:

GGGAGCUCAG AAUAAACGCU CAAACAUACC CGGCGAUCGA AACCCUUAGG 50

UUACAUAAAC CAAUGAGGCC GUCCGGACAC AUAAUUCGAC AUGAGGCCCG 100

GAUCCGGC 108 (2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

GGGAGCUCAG AAUAAACGCU CAACUCCCAA GGCAAUGGAA ACCCUUAGGU 50

UACUACAACC GAUGACGCCA CCCAGGUACU UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

GGGAGCUCAG AAUAAACGCU CAAUGUUCCU GGCAAUAGAA ACCCUUAGGU 50

UAUAAAGACC AAUGAUGCCA UCCGGCUACU UUGUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 104 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

GGGAGCUCAG AAUAAACGCU CAACUCCUGG CAGUAAAAAC CCUUAGGAAG 50

CGAUUCCAAU GAAGCCAUCC GGUUACUUCU UUCGACAUGA GGCCCGGAUC 100

CGGC 104

(2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 104 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

GGGAGCUCAG AAUAAACGCU CAAACAAGGC AAUAGAAACC CUUAGGUUGU 50

UACAACCAAU GAUGCCAUUC GGUCACUUCA UUCGACAUGA GGCCCGGAUC 100

CGGC 104

(2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

GGGAGCUCAG AAUAAACGCU CAACUUCCGA GGCAAUAGAA ACCCUAAGGC 50

UUAAACAACC AAUGAUGCCA UCCAGGCAAG UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

GGGAGCUCAG AAUAAACGCU CAAAAAGCAA GGCUAUCGAA ACCCUAAGGG 50

UGCAAACCCA AUGAGGCCUU UCCGGGAACC UAAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

GGGAGCUCAG AAUAAACGCU CAACUUCCAA GGCAAUAGAA ACCCUUAGGA 50

UACAAGUUCC GAUGAAGCCA CCCGGUCUCG UCAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 108 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

GGGAGCUCAG AAUAAACGCU CAAAAAACAG GACACCAUGA UAAAUGGACG 50

AGUUCACUGG AGCGUCUAAA AGGGCACCCU UGGAUUCGAC AUGAGGCCCG 100

GAUCCGGC 108

(2) INFORMATION FOR SEQ ID NO: 66:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

GGGAGCUCAG AAUAAACGCU CAAUAACAGG ACACCAUGAU UAAUGGACGA 50

GUUCACUAGG GCGGUUAAAA GAGCUCUCGA GGAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO: 67:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

GGGAGCUCAG AAUAAACGCU CAAGAACAGG ACACCAAGAU AAUUGGACGA 50

GUUUACUAGG GCGGCUAUGC UGGCUCUCGA GGAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO: 68:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

GGGAGCUCAG AAUAAACGCU CAAGAACAGG ACACCUGGUU CUCAGGACGA 50

GUUUACUAGG GCGGCAAAAA GGGCUCUCGU GGGUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

GGGAGCUCAG AAUAAACGCU CAAAAACAGG ACACCAAUUU UAUUGGACGA 50

GUUUACUAGG GCGGUAUUAU GGGCUCGCGA GGAUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.-70:

GGGAGCUCAG AAUAAACGCU CAAAAACAGG ACACCAUGAA AAUGGACGAG 50

UUCACUAGAG CGUUGAGCUG UGCGCCCGUG GAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:

GGGAGCUCAG AAUAAACGCU CAAAAACAGG ACACCAAGAU UAUUGGACGA 50

GUUUACUAGG GCGAUUAAUG GGCUCGCGAU GAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 104 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

GGGAGCUCAG AAUAAACGCU CAAAACAGGA CACCUUGAAU AAAGGACGAG 50

UUUACUGUGC GUUUAGUAGA GAACCCGGGA UUCGACAUGA GGCCCGGAUC 100

CGGC 104

(2) INFORMATION FOR SEQ ID NO: 73:

(i) ^'SEQUENCE CHARACTERIZATION: (A) LENGTH: 105 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single.

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:

GGGAGCUCAG AAUAAACGCU CAAGCUCAAG GCAGAAACAG GACACACCAA 50

GACGAGUUAA CCAGCCCAGC UUGACCAUAC AUUCGACAUG AGGCCCGGAU 100

CCGGC 105

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 106 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

GGGAGCUCAG AAUAAACGCU CAAAAGGUAA GAAACAGGAC ACGCACUUAA 50

ACAGACGAGU UAACCAUACC UAGAUCGCGG AAUUCGACAU GAGGCCCGGA 100

UCCGGC 106

(2) INFORMATION FOR SEQ ID NO:75:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 107 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:

GGGAGCUCAG AAUAAACGCU CAAAAAGGCA CUGACCACCC UCAGGAAGAA 50

UAACCGCGGU CACCCGCAUC CGAGUCUAUC AAUUUCGACA UGAGGCCCGG 100

AUCCGGC 107

(2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: WRUNGAAACC CUWAGGNYRN WWVDNCCRAU GANGCC 36

(2) INFORMATION FOR SEQ ID NO:77:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: CGCUCAADAA CAGGACACCW DKWWHWHWGG ACGAGUUYAC URGDGCG 47

(2) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: Xaa

(B) LOCATION: 2

(C) OTHER INFORMATION: AROMATIC OR ALIPHATIC

AMINO ACID (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Phe Xaa Gly Leu Met

5

(2) INFORMATION FOR SEQ ID NO: 79:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: GGCNNNNNNN ACCCUNAGG 19

(2) INFORMATION FOR SEQ ID NO: 80:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: Phe Gly Leu Met 4

(2) INFORMATION FOR SEQ ID NO: 81:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: Arg Pro Lys Pro 4

(2) INFORMATION FOR SEQ ID NO:82:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Pro Gin Gin Phe Phe

5

(2) INFORMATION FOR SEQ ID NO: 83:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: CGUGUCCCCC ACUCGAGAUA UUCGACAUGA GACACG 36 (2) INFORMATION FOR SEQ ID NO: 84:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: GGGAGCUCAG AAUAAACGCU CAACCAGUCU UGUGGCUUUG AAAGAGAGGA 50 GUGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 85:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: GGGAGCUCAG AAUAAACGCU CAACCAGUCU UGUGGCAUUG AAAGAUAGGU 50 GUGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 86:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: GGGAGCUCAG AAUAAACGCU CAACCAGUCU UAUGGCGUUG CAAGAUAGGG 50 GCGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: GGGAGCUCAG AAUAAACGCU CAACCAGUAU UAUGGCUUUG AGAGAGAGGU 50 GCGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:88:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: GGGAGCUCAG AAUAAACGCU CAACCAGUCU UGUGGCUUUG UAAGAGAGGA 50 GUGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 89:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: GGGAGCUCAG AAUAAACGCU CAACCAGUCU UAUGGCUUUG AAAGUGAGGA 50 GUGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:90:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: GGGAGCUCAG AAUAAACGCU CAACGGCACA GGGGUUGUAU CCUCCGGGAC 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:91:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: GGGAGCUCAG AAUAAACGCU CAACGGCACA GGGCCUGUAU CCUCCGGGCC 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:92:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: GGGAGCUCAG AAUAAACGCU CAACGGCAUA GGGGUUGUAU CCUCCGGGAC 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 93:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: GGGAGCUCAG AAUAAACGCU CAACGGCACC GGGGCUGUAU CCUCCGGCAC 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 94:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: GGGAGCUCAG AAUAAACGCU CAAAGAUUGA AUGGGGGUAA CCAACGGGAG 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:95:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: GGGAGCUCAG AAUAAACGCU CAAGUCAAUC AUCGAUGUCC UGUGCCCUAG 50 GGCUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 96:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: GGGAGCUCAG AAUAAACGCU CAAGUCAAUC UUCGAUGUGC UGUGCCCGAU 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 97:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: GGGAGCUCAG AAUAAACGCU CAAGUCAAUC AUCGAUGUGC UGUGCCCGAU 50 AAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 98:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: GGGAGCUCAG AAUAAACGCU CAAGUCAAUU AUCGAUGUGC UGUGCCCGAU 50 CAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 99:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: GGGAGCUCAG AAUAAACGCU CAAGUCAAUU AUCGAUGUGC UGUGCCCGAU 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 100:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: GGGAGCUCAG AAUAAACGCU CAAGUCAAUU AUCAAAGUGC GGAACCCUAU 50 GAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:101:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: GGGAGCUCAG AAUAAACGCU CAAGUCGAGG CCCGGAUGUG CUGUGCCCUG 50 GGAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 102:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: GGGAGCUCAG AAUAAACGCU CAAGUCCUAA UCCCUAAUGU GAUCUGAUGA 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:103:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: GGGAGCUCAG AAUAAACGCU CAAGCCCCCG GUUGAAGACU UGUAAUGCCC 50 UAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:104:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: GGGAGCUCAG AAUAAACGCU CAAGUCUCGC AUUUAGACAG ACCUGUGCCC 50 UAAAUUCGAC AUGAGGCCCG GAUCCGGC 78

(2) INFORMATION FOR SEQ ID NO-.105:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: GGGAGCUCAG AAUAAACGCU CAAUGUUGAG UAAGACGAGC UGUGCCCUUA 50 UUCGACAUGA GGCCCGGAUC CGGC 74

(2) INFORMATION FOR SEQ ID NO:106:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: GGGAGCUCAG AAUAAACGCU CAAUGGUUGU GAAAGAUGAG GUGAGCUCUU 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:107:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: GGGAGCUCAG AAUAAACGCU CAACGCACGA CUAAGGAUGU GCUGUGCCCU 50 UUAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:108:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: GGGAGCUCAG AAUAAACGCU CAAUGUACGA CUAAGCAUGU GCUGUGCCCU 50 UUAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:109:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: GGGAGCUCAG AAUAAACGCU CAAUGGACAC UAGAUGAGGU GCGCUGUGCA 50 CAUUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:110:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: GGGAGCUCAG AAUAAACGCU CAAUUGGAAC UCGAAAUGAU CUGCUGUGCC 50 CAUUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:111:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: GGGAGCUCAG AAUAAACGCU CAAUGUUUGG AGAAGAGCCG UGCCCUCUAG 50 ACAUUCGACA UGAGGCCCGG AUCCGGC 77 (2) INFORMATION FOR SEQ ID NO:112:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: GGGAGCUCAG AAUAAACGCU CAAUGUUUGG AGGAGAGCCG UGCCCUCUAG 50 ACAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:113:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH:76

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: GGGAGCUCAG AAUAAACGCU CAAGUUUGGA GGAGUGAUGU CCUCUCUAGG 50 CAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO: 114:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: GGGAGCUCAG AAUAAACGCU CAAGAAGUGC UGUGCCCUUG ACCGUUUUAU 50 UUCUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO: 115:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: GGGAGCUCAG AAUAAACGCU CAAGAUGUGC UGUGCCCUUG AGUCGUUUCC 50 AGUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:116:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GGGAGCUCAG AAUAAACGCU CAAGAUGUGC UGUGCCCUUC CUCCGUUUCC 50 AAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO: 117:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: GGGAGCUCAG AAUAAACGCU CAAGAUGUGC UGUGCCCUUG GCCAGUUUCC 50 AAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:118:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: GGGAGCUCAG AAUAAACGCU CAAUCGGUAU GUGCUGUGCC CCCGAGAGUU 50 CGUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO: 119:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: GGGAGCUCAG AAUAAACGCU CAAGCGGAUG UGCGGUGCCC UGCUUAAACG 50 UUGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:120:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: GGGAGCUCAG AAUAAACGCU CAAGCGCUGC CUCAGGUAAU GCCCUUAGAA 50 AGUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:121:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: GGGAGCUCAG AAUAAACGCU CAAGCGAUCG ACUGCAUCAU AUGGCACGAG 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:122:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: GGGAGCUCAG AAUAAACGCU CAAGUGGUGA AUCAGUGCGU GUGUGGCCUA 50 GAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:123:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single -

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: GGGAGCUCAG AAUAAACGCU CAAUGUCCGA AAAUCACGUU GCUGCAGACA 50 CAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO: 124:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: GGGAGCUCAG AAUAAACGCU CAAACAUCGA UGACCGGAAU GCCGCACACA 50 GAGUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:125:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: GGGAGCUCAG AAUAAACGCU CAAUAAGCCU CACGUUUGUC UGAACAGGAU 50 CGUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:126:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: GGGAGCUCAG AAUAAACGCU CAAGCCUCAC UGUUGUAUUG UGCCGCAUGG 50 CAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO: 127:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: GGGAGCUCAG AAUAAACGCU CAAUCCAUGU UCGAUAUACA GGAUGGAAAG 50 GUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:128:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) -SEQUENCE DESCRIPTION: SEQ ID NO: 128: GGGAGCUCAG AAUAAACGCU CAAUGUCCUU AACUUGCUAC UUCACGCUGU 50 ACUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:129:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: GGGAGCUCAG AAUAAACGCU CAAUGUCCGU UUUAUGUCAA AUGUAUUUCG 50 UAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:130:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: GGGAGCUCAG AAUAAACGCU CAAGAUCCGC AGUAACUGAU AAUGUUAAAG 50 UACUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:131:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: GGGAGCUCAG AAUAAACGCU CAAUAGCCGG GUCAAGAAAG CCGGACAGUG 50 UUAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:132:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: GGGAGCUCAG AAUAAACGCU CAAGAGGCUC AACCCUUACU GCAUGCUGGU 50 CAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:133:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: GGGAGCUCAG AAUAAACGCU CAAGUUCACA AGAGGAAACC AUUAAUGCUA 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO: 134:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: GGGAGCUCAG AAUAAACGCU CAACGACGCU AAACGUAGCU UGGUUGUGUA 50 UUCGACAUGA GGCCCGGAUC CGGC 74

(2) INFORMATION FOR SEQ ID NO:135:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: GGGAGCUCAG AAUAAACGCU CAAUGACUAU GGGCUAGACU GCUUGGUGAA 50 UUCGACAUGA GGCCCGGAUC CGGC 74

(2) INFORMATION FOR SEQ ID NO: 136:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 76 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136: GGGAGCUCAG AAUAAACGCU CAAACCCCUG ACGCGCACGU AUAGCUGACU 50 AAUUCGACAU GAGGCCCGGA UCCGGC 76

(2) INFORMATION FOR SEQ ID NO:137:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: GGGAGCUCAG AAUAAACGCU CAACCUGAGA ACUGAAGCCC UCGUCUGCCG 50 UAAUUCGACA UGAGGCCCGG AUCCGGC 77

(2) INFORMATION FOR SEQ ID NO:138:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: GGGAGCUCAG AAUAAACGCU CAAGUCGACA CGUACUGAGG UCGCGGAAGU 50 AUUCGACAUG AGGCCCGGAU CCGGC 75

(2) INFORMATION FOR SEQ ID NO:139:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: CAATGACCGC ATGGGATCCG TGTGGAAAAT CTCTAGCAGT 40 (2) INFORMATION FOR SEQ ID NO:140:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 77 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: GCCGGATCCG GGCCTCATGT CGAANNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNTTGAGC GTTTATTCTG AGCTCCC 77

(2) INFORMATION FOR SEQ ID NO: 141:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: CCGAAGCTTA ATACGACTCA CTATAGGGAG CTCAGAATAA ACGCTCAA 48

(2) INFORMATION FOR SEQ ID NO: 142:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142: GCCGGATCCG GGCCTCATGT CGAA 24

(2) INFORMATION FOR SEQ ID NO:143:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143: CCCGGATCCT CTTTACCTCT GTGTG 25

(2) INFORMATION FOR SEQ ID NO:144:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: CCGAAGCTTA ATACGACTCA CTATAGGGAC TATTGATGGC CTTCCGACC 49

(2) INFORMATION FOR SEQ ID NO:145:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: CCCCTGCAGG TGATTTTGCT CAAGTNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN AGTATCGCTA ATCAGGCGGA T 81 (2) INFORMATION FOR SEQ ID NO:146:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: ATCCGCCTGA TTAGCGATAC T 21

(2) INFORMATION FOR SEQ ID NO: 147:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(A) NAME/KEY: N

(B) LOCATION: 1

(C) OTHER INFORMATION: This symbol stands for biotintylated cytosine.^'

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: NCCCTGCAGG TGATTTTGCT CAAGT 25

(2) INFORMATION FOR SEQ ID NO: 148:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:148: CCGAAGCTTA ATACGACTCA CTATAGGGAT CCGCCTGATT AGCGATACT 49

(2) INFORMATION FOR SEQ ID NO:149:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:149: TTCACACAGG AAACAG 16

(2) INFORMATION FOR SEQ ID NO:150:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:150: ATCCGCCTGA TTAGCGATAC TCAGGCTCCT GAGTGAAGTG CGGACATGTA 50 CCNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 151:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151: ATCCGCCTGA TTAGCGATAC TCGCCAGGCC CCTGTAGTCG GGCGGAGTCA 50 NNNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:152:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 82 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152: ATCCGCCTGA TTAGCGATAC TCGTATAGGT CCCCTGCCGC TAAACAGCGC 50 CGCGGTAACT TGAGCAAAAT CACCTGCAGG GG ^' 82

(2) INFORMATION FOR SEQ ID NO:153:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:153: ATCCGCCTGA TTAGCGATAC TCTGCCAGTC CCCTGTAATT AGACGGAAAC 50 TCCTGTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:154:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:154: ATCCGCCTGA TTAGCGATAC TCAGCAGTCC CCCTATTCAT GGGCCCGCGG 50 TTCATGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:155:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: ATCCGCCTGA TTAGCGATAC TTAACGCCAG GCCCCTGTAA TAGTGCGGAT 50 CGACAGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:156:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:156: ATCCGCCTGA TTAGCGATAC TGAGCTGTTG TACAGTGCAA GTGTAGCAGT 50 TCCCCTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 157: (i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157: ATCCGCCTGA TTAGCGATAC TGTATCTTTA GTACAAGTGC TCGGCAGCTC 50 CCCCACACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:158:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158: ATCCGCCTGA TTAGCGATAC TTCGCCAGTC CCCTGTTTCA GCGCGGATAT 50 GACCATACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 159:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: ATCCGCCTGA TTAGCGATAC TGTATGGCTC TCAGCCCAGG CCCCTGATAC 50 AGTCGACTTG AGCAAAATCA CCTGCAGGGG 80

(2) INFORMATION FOR SEQ ID NO:160:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: ATCCGCCTGA TTAGCGATAC TGAAGAGCGT GCTGTCCCCT TAGGGTAATT 50 GTCNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:161:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:161: ATCCGCCTGA TTAGCGATAC TACGCGTGCT GCCCCATAAC GGTGGCTTCA 50 ANNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:162:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162: ATCCGCCTGA TTAGCGATAC TGACAATGAG TCAAGTCGCG TGCTCCCCTG 50 CTGTTGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:163:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:163: ATCCGCCTGA TTAGCGATAC TCGGGCCCCT GATTAACGCG CGCTGCCCCT 50 CGGGTGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 164:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:164: ATCCGCCTGA TTAGCGATAC TCGATATGAG CGTGAGCGTG CTTCCCTTGT 50 TGGTGNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 165:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165: ATCCGCCTGA TTAGCGATAC TGTCTGTCAG ATTCATGCGT GCTCCCCCTT 50 CTGGTGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:166:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166: ATCCGCCTGA TTAGCGATAC TCTGGAGCGT GCTGCCCCTA AAGGTGACTT 50 ACCAAGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 167:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167: ATCCGCCTGA TTAGCGATAC TTAGCTACAC TATATGGCGT GCTCCCCCTG 50 TTCGTGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 168:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 81 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168: ATCCGCCTGA TTAGCGATAC TCTTGGCCCG TATTCGCGTG CTGTCCCCCT 50 GAGATGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:169:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169: ATCCGCCTGA TTAGCGATAC TGAACGTGCA GCCCCCCGAA ACGTGACTAG 50 CAANNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:170:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170: ATCCGCCTGA TTAGCGATAC TGGATTTTTG TGCAAGCCCC CGAAAGCTGA 50 TNNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 171:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:171: ATCCGCCTGA TTAGCGATAC TACGTCAGGA CCCCTCATCG ATTTTCTTAA 50 GNNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:172:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:172: ATCCGCCTGA TTAGCGATAC TTTAGCAAAG GAGCCCCCGG ACTCAGATTA 50 CNNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:173:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:173: ATCCGCCTGA TTAGCGATAC TTGTTATAGT CCCCTGCCGC TGTTCTCGCG 50 GGATTNACTT GAGCAAAATC ACCTGCAGGG G 81 (2) INFORMATION FOR SEQ ID NO:174:

(i) SEQUENCE CHARACTERIZATION: .

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:174: ATCCGCCTGA TTAGCGATAC TCAAGTCAAA TCCCCTGACA GGAATTCCTT 50 CCTGGAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 175:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175: ATCCGCCTGA TTAGCGATAC TTGTTCAGTC CCCCTCTCAA GCTACTTTAC 50 TTTGTAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 176:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:176: ATCCGCCTGA TTAGCGATAC TAGCGAGCTT ATTAGAAGGA TAAACCGCCT 50 ANNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 177:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:177: ATCCGCCTGA TTAGCGATAC TTGCTGGTCA TAGGTAAACA GCCCTGAGCT 50 AACAGAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:178:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCCA GAACATGGAA 50 TATATCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 179:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179: ATCCGCCTGA TTAGCGATAC TATCGAGGTG ATCAGAAGGA TAAACCGCCG 50 GGGCCTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:180:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 180: ATCCGCCTGA TTAGCGATAC TCTAAACGGT GAAGGGTCTT TGCAGATGAA 50 CAANNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 181:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:181: ATCCGCCTGA TTAGCGATAC TTTAGCAAAG TAGAAGCCGG TTAGAAGACC 50 TAGAACACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:182:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:182: ATCCGCCTGA TTAGCGATAC TTTAGCAAAG TTGAAGCCGG ACTAACAAGC 50 TCTACGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:183:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 79 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:183: ATCCGCCTGA TTAGCGATAC TGGGCTCAAG CTTGAGCGCG GCTCTCCACC 50 TACGACTTGA GCAAAATCAC CTGCAGGGG 79

(2) INFORMATION FOR SEQ ID NO: 184:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:184: ATCCGCCTGA TTAGCGATAC TTGTCGGGTG GCTTTAGCAG AGACAATATG 50 CATTNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 185:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 185: ATCCGCCTGA TTAGCGATAC TCTATAACCA GGTTTCGGGT GCTTTAGCAA 50 ANNNNNACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 186:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:186: ATCCGCCTGA TTAGCGATAC TGGGAGGGAG GGAGGGCCGT AGCTAATTAG 50 GATCAAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:187:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:187: ATCCGCCTGA TTAGCGATAC TACGCGTGCT GCCCCTAAAG GCGATTGTCG 50 GATGTTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 188:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:188: ATCCGCCTGA TTAGCGATAC TTACGTGAGC GTGCTGTCCC CTAAAGGTGA 50 TACGTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:189:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:189: ATCCGCCTGA TTAGCGATAC TCTGGAGCGT GCTGCCCCTA AAGGTGACTT 50 ACCAAGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:190:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:190: ATCCGCCTGA TTAGCGATAC TCGCGTGCTG CCCCTTAAGG TGATGGTGTA 50 TATTCCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:191:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 79 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191: ATCCGCCTGA TTAGCGATAC TTCTCCGACT CAAAGCGCGT GCTCCCCTCC 50 GGTGACTTGA GCAAAATCAC CTGCAGGGG 79

(2) INFORMATION FOR SEQ ID NO: 192:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 82 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:192: ATCCGCCTGA TTAGCGATAC TCGTATAGGT CCCCTGCCGC TAAACAGCGC 50 CGCGGTAACT TGAGCAAAAT CACCTGCAGG GG 82

(2) INFORMATION FOR SEQ ID NO:193:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 193: ATCCGCCTGA TTAGCGATAC TGCCAGGTCC CCTGTAATTA GACGGAAACT 50 ACCTGTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:194:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:194: ATCCGCCTGA TTAGCGATAC TGCCAGGACC CCTGTAATCT GGCGTATTTC 50 CCTGTTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:195:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-.195: ATCCGCCTGA TTAGCGATAC TCGCCAGTAC CCCTGTAAGT GGGCGGAAAC 50 TCTAGTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:196:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 196: ATCCGCCTGA TTAGCGATAC TTCGTCAGGA CCCCTGTAAA CAGGCGGGAT 50 AATCTAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO: 197:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 197: ATCCGCCTGA TTAGCGATAC TGGGCCCTCA GCTTGAGCGC GGACTACATA 50 TTATCACTTG AGCAAAATCA CCTGCAGGGG 80

(2) INFORMATION FOR SEQ ID NO:198:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:198: ATCCGCCTGA TTAGCGATAC TGGGCCCTCA GCTTGAGCGC GGAATCACTA 50 AGATACACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:199:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 199: ATCCGCCTGA TTAGCGATAC TGGGCCCTCA GCTAGAGCCG GATTAAACAG 50 TCTTCAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:200:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:200: ATCCGCCTGA TTAGCGATAC TTATTTGCCC TTGCAGGCCG CAGGAGTGCT 50 AGCAGTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO-.201:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-.201: ATCCGCCTGA TTAGCGATAC TCAGGCGTTA GGGAAGGGCG TCGAAAGCAG 50 GGTGGGACTT GAGCAAAATC ACCTGCAGGG G^' 81 (2) INFORMATION FOR SEQ ID NO:202:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:202: ATCCGCCTGA TTAGCGATAC TCAGGCGCCG GGGGGGTGGG AATACAGTGA 50 TCAGCGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:203:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:203: ATCCGCCTGA TTAGCGATAC TCAGGCCTTG GGCGGGCCGG GACAATGGAG 50 AGATTTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:204:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:204: ATCCGCCTGA TTAGCGATAC TAGCCAGTCA AGTTAATGGG TGCCATGCAG 50 AAGCAACTTG AGCAAAATCA CCTGCAGGGG 80

(2) INFORMATION FOR SEQ ID NO:205:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:205: ATCCGCCTGA TTAGCGATAC TAATCGGCCT TGTTTCGGGG TGCTTTAGCA 50 GAGGAAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:206:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:206: ATCCGCCTGA TTAGCGATAC TCAGGGTGCC GCTCAATTCT GGGTGCCTTG 50 CAGAAGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:207:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 79 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207: ATCCGCCTGA TTAGCGATAC TCCAGCGGTG GCATCACGCG GACTTACTCT 50 AGCAACTTGA GCAAAATCAC CTGCAGGGG 79

(2) INFORMATION FOR SEQ ID NO:208:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:208: ATCCGCCTGA TTAGCGATAC TTTAGCAAAG TTGAAGCCGG ACTAACAAGC 50 TCTACGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:209:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:209: ATCCGCCTGA TTAGCGATAC TCTAGCAGAG TAGAAGCCGG ACGATATATC 50 GATGATACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:210:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:210: ATCCGCCTGA TTAGCGATAC TGGACTCCCA GTTGATGCGC GGTCTTTATC 50 ACCTCCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:211:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:211: ATCCGCCTGA TTAGCGATAC TAAGCTCTTA GTTGATGCGC GGTCAAAATT 50 TAAGCTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:212:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:212: ATCCGCCTGA TTAGCGATAC TGAAGCTCTT TTAGTGATGC GTGGACCAGT 50 CCCCTTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:213:

(i) SEQUENCE CHARACTERIZATION: (A) LENGTH: 81 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:213: ATCCGCCTGA TTAGCGATAC TGGGCTCCAG CTTGAGCGGC GACTTAATTG 50 GTTATTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:214:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 80 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:214: ATCCGCCTGA TTAGCGATAC TGATATACTT ATTACTTCGC ACGGCTAACC 50 AGACCACTTG AGCAAAATCA CCTGCAGGGG 80

(2) INFORMATION FOR SEQ ID NO:215:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:215: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCCA GAACTTGGAA 50 TATATCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:216:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:216: ATCCGCCTGA TTAGCGATAC TCTCGAGGTG ATCAGAAGGA TAAACCGCCG 50 GGGCCTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:217:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:217: GGUCCGAAGU GCAACGGGAA AAUGCACU 28

(2) INFORMATION FOR SEQ ID NO:218:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:218: GTCCCTGTTC GGGCGCCA 18

(2) INFORMATION FOR SEQ ID NO:219: (i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:219: CAGGGACAAG CCCGCGGUGA CGAUCUCUAA 30

(2) INFORMATION FOR SEQ ID NO:220:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:220: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT ATAGTGTCTA CAACTACGGC 50 TACGTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:221:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:221: ATCCGCCTGA TTAGCGATAC TCAGACGGCG AGTCGGCCTA GCACGTGGAC 50 GATTTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:222:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:222: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT ATACTGTCTA GAACTTGGAA 50 AGTGTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:223:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:223: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACCGTCCG GGACTTGCAA 50 TGAATAACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:224:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) ^• SEQUENCE DESCRIPTION: SEQ ID NO:224: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT AAACTGTCTA GAACTTGGAG 50 TCCATCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:225:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:225: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT ACACTGTCTA GAACCTAGAG 50 TACGTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:226:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:226: ATCCGCCTGA TTAGCGATAC TCAGGAGGAA CGACGGGACA GACCTTGGCA 50 TGTAGCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:227:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:227: ATCCGCCTGA TTAGCGATAC TCAGTCGGCC AAACTGTGAA GAACTCGGAC 50 GCCCTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:228:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:228: ATCCGCCTGA TTAGCGATAC TCCGGAGGCT CAACTGTCCA GCAATTCGCA 50 CTCATCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:229:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:229: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT AAACTGTCTA GAACCACGAA 50 TTTCCCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:230:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:230: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AGGCTGCCTA GAGCTTGGAA 50 TTTAGGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:231:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:231: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT AAACAGCCCT GAGCTTGGAA 50 GTCGTCACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:232:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:232: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCTA GAACTTGGAA 50 TATATTACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:233:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:233: ATCCGCCTGA TTAGCGATAC TCGGAAGGAT AAAGTGCCCA CAGCCTGGAA 50 TGTAACACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:234:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:234: ATCCGCCTGA TTAGCGATAC TCAGTAGGAT AAACTGTCTA GAACGCGGAA 50 GATATGACTT GAGCAAAATC ACCTGCAGGG G 81

(2) INFORMATION FOR SEQ ID NO:235:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 81 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:235: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCGA GAACCTCGAA 50 TATGTCACTT GAGCAAAATC ACCTGCAGGG G 81 (2) INFORMATION FOR SEQ ID NO:236:

(i) SEQUENCE CHARACTERIZATION: .

(A) LENGTH: 56 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:236: ATCCGCCTGA TTAGCGATAC TCGGNAGGAN ANNCNGNNTN GNNCNNNGNN 50 NNCNNN 56

(2) INFORMATION FOR SEQ ID NO:237:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:237: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCCA GAACTTGGA 49

(2) INFORMATION FOR SEQ ID NO:238:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:238: ATCCGCCTGA ATAGCGATAC TCAGAAGGAT 30

(2) INFORMATION FOR SEQ ID NO:239:

(i) SEQUENCE CHARACTERIZATION:

(A) LENGTH: 53 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ix) FEATURE:

(D) OTHER INFORMATION: The four 3' thymine residues are linked by a phosphorothioate backbone (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239: ATCCGCCTGA TTAGCGATAC TCAGAAGGAT AAACTGTCCA GAACTTGGAT 50 TTT 53

Claims

CLAIMS :

1. A method of identifying nucleic acid ligands to insulin receptor antibodies, comprising: a) preparing a candidate mixture of nucleic acids; b) contacting a candidate mixture of nucleic acids with insulin receptor antibodies, wherein nucleic acids having an increased affinity to insulin receptor antibodies relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to insulin receptor antibodies may be identified.

2. The method of claim 1 further comprising: e) repeating steps b) , c) , and d) .

3. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

4. The method of claim 3 wherein said single stranded nucleic acids are ribonucleic acids.

5. The method of claim 3 wherein said nucleic acids are modified nucleic acids.

6. The method of claim 5 wherein said nucleic acids are 2'-amino (2'-NH₂) modified ribonucleic acids.

7. A nucleic acid ligand to insulin receptor antibodies identified according to the method of claim 1.

8. A purified and isolated non-naturally occurring RNA ligand to an insulin receptor antibody.

9. The RNA ligand of claim 8 wherein said ligand is selected from the group consisting of the sequences set forth in Figure 2 (SEQ ID NOS:4-15) .

10. The RNA ligand of claim 9 wherein said ligand is substantially homologous to and has substantially the same ability to bind the insulin receptor antibody as^' a ligand selected from the group consisting of the sequences set forth in Figure 2 (SEQ ID NOS:4-15) , or the corresponding DNA sequences thereof or the corresponding complementary sequences thereof.

11. The RNA ligand of claim 9 wherein said ligand has substantially the same structure and substantially the same ability to bind the insulin receptor antibody as a ligand selected from the group consisting of the sequences set forth in Figure 2 (SEQ ID NOS:4-15), or the corresponding DNA sequences thereof or the corresponding complementary sequences thereof.

12. The RNA ligand of claim 9 wherein said ligand has been chemically modified at the ribose and/or phosphate and/or base positions.

13. The RNA ligand of claim 12 wherein said ligand is comprised of 2' -amino (2'-NH₂) modified nucleotides.

14. The RNA ligand of claim 7 wherein said insulin receptor antibody targets a human insulin receptor.

15. The RNA ligand of claim 14 wherein said insulin receptor antibody targets amino acids residues 450-601 on the α subunit of said human insulin receptor.

16. The RNA ligand of claim 15 wherein said insulin receptor antibody is an autoantibody.

17. The RNA ligand of claim 15 wherein said insulin receptor antibody is a monoclonal antibody.

18. The RNA ligand of claim 17 wherein said insulin receptor antibody is MA-20.

19. The RNA ligand of claim 16 wherein said ligand is an inhibitor of autoantibody binding to said human insulin receptor.

20. The RNA ligand of claim 17 wherein said ligand is an inhibitor of monoclonal antibody binding to said human insulin receptor.

21. The RNA ligand of claim 18 wherein said ligand is an inhibitor of MA-20 binding to said human insulin receptor.

22. A method for treating Type B insulin resistance comprising administering a pharmaceutically effective amount of a nucleic acid insulin receptor antibody ligand.

23. The method of claim 22 wherein said nucleic acid insulin receptor antibody is identified according to the method of claim 1.

24. The method of claim 23 wherein said ligand is selected from one of the ligands of Figure 2 (SEQ ID NOS:4-15) .

25. A method of identifying nucleic acid ligands to human antibodies, comprising: a) preparing a candidate mixture of nucleic acids ; b) contacting a candidate mixture of nucleic acids with said human antibodies, wherein nucleic acids having an increased affinity to the human antibodies relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to said human antibodies may be identified.

26. The method of claim 25 further comprising: e) repeating steps b) , c) , and d) .

27. The method of claim 25 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

28. The method of claim 25 wherein said single stranded nucleic acids are ribonucleic acids.

29. The method of claim 25 wherein said nucleic acids are modified nucleic acids.

30. The method of claim 25 wherein said nucleic acids are 2' -amino (2'-NH₂) modified ribonucleic acids.

31. The method of claim 25 wherein said human antibodies recognize complex tertiary molecular structures.

32. The method of claim 25 wherein said human antibodies are human insulin receptor antibodies.

33. A method of identifying nucleic acid ligands to antibodies or other proteins (termed protein A) which bind to some other protein(s) (termed protein B) where the identified nucleic acid mimics the structure of protein B and interacts with protein A in an analogous way as protein B. The said method comprises: a) preparing a candidate mixture of nucleic acids; b) contacting a candidate mixture of nucleic acids with said antibody or protein A, wherein nucleic acids having an increased affinity to said antibody or protein A and which mimic the structure of protein B and its interaction with said antibody or protein A relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids which mimic protein B from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids which mimic protein B to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to said antibody or protein A may be identified.

34. The method of claim 33 further comprising: e) repeating steps b) , c) , and d) .

35. The method of claim 33 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

36. The method of claim 33 wherein said single stranded nucleic acids are ribonucleic acids.

37. The method of claim 33 wherein said nucleic acids are modified nucleic acids.

38. The method of claim 33 wherein said nucleic acids are 2' -amino (2'-NH₂) modified ribonucleic acids.

39. A method of identifying nucleic acid ligands to Substance P (SP) comprising: a) preparing a candidate mixture of nucleic acids; b) contacting a candidate mixture of nucleic acids with SP, wherein nucleic acids having an increased affinity to the SP relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to SP may be identified.

40. The method of claim 39 further comprising: e) repeating steps b) , c) and d) .

41. The method of claim 39 further comprising: e) contacting, between steps c) and d) , the increased affinity nucleic acids with one or more non- substance P molecules, wherein nucleic acids with an affinity to the non-Substance P molecules (s) is removed.

42. The method of claim 39 further comprising: e) preparing a second candidate mixture of nucleic acids by mutagenizing the ligand-enriched mixture of nucleic acids; and f) repeating steps b) , c) , and d) , whereby nucleic acid ligands of SP may be identified.

43. The method of claim 39 wherein said candidate mixture of nucleic acids is comprised of single- stranded nucleic acids.

44. The method of claim 43 wherein said single- stranded nucleic acids are ribonucleic acids.

45. The method of claim 43 wherein said single- stranded nucleic acids are deoxyribonucleic acids .

46. A nucleic ligand to SP, identified according to the method of claim 39.

47. A purified and isolated non-naturally occurring

RNA ligand to SP, wherein said ligand is selected from the group consisting of the sequences set forth in Figures 9 (SEQ ID NOS:26-42) and 10 (SEQ ID NOS:43-64) , or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof .

48. The RNA ligand of claim 47 wherein said ligand is selected from the group consisting of SEQ ID NOS:22, 35-38, 43-64 and 75.

49. A purified and isolated non-naturally occurring RNA ligand to SP, wherein said ligand is substantially homologous to and has substantially the same ability to bind SP as a ligand selected from the group consisting of the sequences set forth in Figures 9 (SEQ ID NOS:26- 42) and 10 (SEQ ID NOS:43-64) , or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof.

50. A purified and isolated non-naturally occurring

RNA ligand to SP, wherein said ligand has substantially the same structure and substantially the same ability to bind SP as a ligand selected from the group consisting of the sequences set forth in Figures 9 (SEQ ID NOS:26-42) and 10 (SEQ ID NOS:43-64) , or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof.

51. The RNA ligand of claim 47 wherein said ligand has been chemically modified at the ribose and/or phosphate and/or base positions.

52. The modified RNA ligand of claim 51 wherein said ligand is comprised of 2' -amino (2'-NH₂) modified nucleotides.

53. A method for identifying nucleic acid ligands from a candidate mixture of nucleic acids, said nucleic acid ligands being a ligand of a given target molecule comprising: a) contacting said candidate mixture with the target molecule, wherein nucleic acids having increased affinity to the target molecule may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; d) preparing a second candidate mixture of nucleic acids by mutagenizing the ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule may be identified.

54. The method of claim 53 further comprising: e) repeating steps a) , b) , and c) .

55. A method of identifying nucleic acid ligands and nucleic acid ligand sequences to a target molecule comprising: a) preparing a candidate mixture of nucleic acids; b) contacting a candidate mixture with the target molecule, wherein nucleic acids having an increased affinity to the target molecule relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand enriched mixture of nucleic acids whereby nucleic acid ligands of the target molecule may be identified.

56. The method of claim 55, further comprising: e) repeating steps b) , c) , and d) .

57. The method of claim 55, wherein the candidate mixture contacted includes non-amplifiable random pool nucleic acids.

58. A method of identifying nucleic acid ligands to HIV integrase, comprising: a) preparing a candidate mixture of nucleic acids; b) contacting a candidate mixture of nucleic acids with HIV integrase, wherein nucleic acids having an increased affinity to the HIV integrase relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; and c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to HIV integrase, whereby nucleic acid ligands of HIV integrase may be identified.

59. The method of claim 58 further comprising: e) repeating steps b) , c) and d) .

60. The method of claim 58, wherein the candidate mixture contacted includes non-amplifiable random pool nucleic acids.

61. The method of claim 58 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

62. The method of claim 61 wherein said single stranded nucleic acids are ribonucleic acids.

63. The method of claim 61 wherein said nucleic acids are modified nucleic acids.

64. The method of claim 63 wherein said nucleic acids are 2'-amino (2'-NH₂) modified ribonucleic acids.

65. The method of claim 58, wherein said HIV integrase is HIV-1 integrase.

66. The method of claim 60, wherein said HIV integrase is HIV-1 integrase.

67. A nucleic acid ligand to HIV-1 integrase identified according to the method of claim 65.

68. A nucleic acid ligand to HIV-1 integrase identified according to the method of claim 66.

69. A purified and isolated non-naturally occurring RNA ligand to HIV-1 integrase, wherein said ligand is selected from the group consisting of the sequences set forth in Table 4, or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof.

70. A purified and isolated non-naturally occurring RNA ligand to HIV-1 integrase, wherein said ligand is substantially homologous to and has substantially the same ability to bind HIV-1 integrase as a ligand selected from the group consisting of the sequences set forth in Table 4, or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof.

71. A purified and isolated non-naturally occurring RNA ligand to HIV-1 integrase, wherein said ligand has substantially the same structure and substantially the same ability to bind HIV-1 integrase as a ligand selected from the group consisting of the sequences set forth in Table 4, or the corresponding DNA sequence thereof or the corresponding complementary sequences thereof.

72. The RNA ligand of claim 69 wherein said ligand has been chemically modified at the ribose and/or phosphate and/or base positions.

73. The modified RNA ligand of claim 72 wherein said ligand is comprised of 2'-amino (2'-NH₂) modified nucleotides.

74. The RNA ligand of claim 67 wherein said ligand is an inhibitor of HIV-1 integrase.

75. The RNA ligand of claim 68 wherein said ligand is an inhibitor of HIV-1 integrase.

76. A purified and isolated non-naturally occurring nucleic acid ligand to HIV-1 integrase identified according to the method of claim 58.

77. A purified and isolated non-naturally occurring nucleic acid ligand to HIV-1 integrase identified according to the method of claim 60.

78. A method for treating HIV comprising administering a pharmaceutically effective amount of a nucleic acid HIV integrase ligand.

79. A method for treating HIV type 1 comprising administering a pharmaceutically effective amount of a nucleic acid HIV-1 integrase ligand.

80. The method of claim 78 wherein said nucleic acid HIV integrase ligand is identified according to the method of claim 58.

81. The method of claim 78 wherein said nucleic acid HIV integrase ligand is identified according to the method of claim 60.

82. The method of claim 79 wherein said nucleic acid HIV-1 integrase ligand is identified according to the method of claim 58.

83. The method of claim 79 wherein said nucleic acid HIV-1 integrase ligand is identified according to the method of claim 60.

84. The method of claim 83 wherein said ligand is selected from one of the ligands of Table 4.

85. A method of identifying nucleic acid ligands and ligand sequences to HIV-1 reverse transcriptase, comprising: a) preparing a candidate mixture of deoxyribonucleic acids (DNA) ; b) contacting the candidate mixture of DNA with

HIV-1 reverse transcriptase, wherein nucleic acids having an increased affinity to HIV-1 reverse transcriptase relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to HIV-1 reverse transcriptase may be identified.

86. The method of claim 85 further comprising: e) repeating steps b) , c) and d) .

87. The method of claim 85 wherein said candidate mixture of nucleic acids is comprised of single stranded DNA.

88. A deoxyribonucleic acid ligand to HIV-1 reverse transciptase identified according to the method of claim 85.

89. A purified and isolated non-naturally occurring DNA ligand to HIV-1 reverse transcriptase.

90. The DNA ligand of claim 89 wherein said ligand is selected from the group consisting of the sequences set forth in Figure 18 (SEQ ID NOS:150-186) .

91. The DNA ligand of claim 89 wherein said ligand is selected from the group consisting of the sequences set forth in Figure 19 (SEQ ID NOS:187-216) .

92. The DNA ligand of claim 89 wherein said ligand is an inhibitor of HIV-1 RT.

93. The DNA ligand of claim 90 wherein said ligand is substantially homologous to and has substantially the same ability to bind HIV-1 reverse transcriptase as a ligand selected from the group consisting of the sequences set forth in Figure 18 (SEQ ID NOS:150-186) .

94. The DNA ligand of claim 91 wherein said ligand is substantially homologous to and has the same ability to bind HIV-1 reverse transcriptase as a ligand selected from the group consisting of the sequences set forth in Figure 19 (SEQ ID NOS:187-216) .

95. The DNA ligand of claim 90 wherein said ligand has substantially the same structure and substantially the same ability to bind HIV-1 reverse transcriptase as a ligand selected from the group consisting of the sequences set forth in Figure 18 (SEQ ID NOS:150-186) .

96. The DNA ligand of claim 91 wherein said ligand has substantially the same structure and substantially the same ability to bind HIV-1 reverse transcriptase as a ligand selected from the group consisting of the sequences set forth in Figure 18 (SEQ ID NOS:150-186) .

97. The DNA ligand of claim 90 wherein said ligand has been chemically modified at the deoxyribose and/or phosphate and/or base positions.

98. The DNA ligand of claim 91 wherein said ligand has been chemically modified at the deoxyribose and/or phosphate and/or base positions.

99. The DNA ligand of claim 90 wherein a nucleotide analog possessing a reactive group which is able to crosslink the ligand to HIV-1 RT has been incorporated.

100. The DNA ligand of claim 99 wherein said nucleotide analog is incorporated to said ligand at the 3'-end.

101. The DNA ligand of claim 100 wherein the covalent crosslinking is coupled to the activity of HIV-1 RT.

102. The DNA ligand of claim 91 wherein a nucleotide analog possessing a reactive group which is able to crosslink the ligand to HIV-1 RT has been incorporated.

103. The DNA ligand of claim 102 wherein said nucleotide analog is incorporated to said ligand at the 3' -end.

104. The DNA ligand of claim 103 wherein the covalent crosslinking is coupled to the activity of HIV-1 RT.

105. A purified and isolated non-naturally occurring nucleic acid ligand to HIV-1 reverse transcriptase identified according to the method of claim 85.

106. A method for treating HIV type 1 comprising administering a pharmaceutically effective amount of a DNA HIV-1 reverse transcriptase ligand.

107. The method of claim 106 wherein said DNA HIV-1 reverse transcriptase ligand is identified according to the method of claim 85.

108. The method of claim 107 wherein said ligand is selected from one of the ligands of Figure 18 (SEQ ID NOS:150-186) .

109. The method of claim 107 wherein said ligand is selected from one of the ligands of Figure 19 (SEQ ID NOS:187-216) .