US005756291A United States Patent (19) 11) Patent Number: 5,756,291 Griffin et al. (45) Date of Patent: May 26, 1998

54 APTAMERS SPECIFIC FOR Ellington and Szostak, "In Vitro Selection of RNA Mol BIOMOLECULES AND METHODS OF ecules that Bind Specific Ligands." Nature (1990) MAKING 346:818-822. Thiesen and Bach, "Target Detection Assay (TDA): A Ver 75 Inventors: Linda Griffin, Atherton; Glenn satile Procedure to Determine DNA Binding Sites as Dem Albrecht, Redwood City; John onstrated on SP1. Protein." Nucleic Acids Res. (1990) Latham, Palo Alto; Lawrence Leung, 18(11):3203-3208. Hillsborough; Eric Vermaas, Oakland; Springer, T.A.. "Adhesion Receptors of the Immune Sys John J. Toole, Burlingame, all of Calif. tem.” Nature (1990) 346:425-434. Isobe. M., et al. "Specific Acceptance of Cardiac Allograft 73 Assignee: Gilead Sciences, Inc.. Foster City, After Treatment with Antibodies to ICAM-1 and LFA-1." Calif. Science (1992) 255:425-434. Oppenheimer-Marks et al., “Differential Utilization of 21 Appl. No.: 484,192 ICAM-1 and VCAM-1. During the Adhesion and Transen 22 Filed: Jun. 7, 1995 dothelial Migration of Human T Lymphocytes." Immunol, (1991) 147:2913-2921. Related U.S. Application Data Straunton et al., “The Arrangement of the Immunoglobulin like Domains of ICAM-1 and the Binding Sites for LFA-1 63 Continuation of Ser. No. 934,387, Aug. 21, 1992, aban and Rhinovirus." Cell (1990) 61:243-254. doned, which is a continuation-in-part of PCT/US92/01383, Ferns et al., "Inhibition of Neointimal Smooth Muscle Feb. 21, 1992. Accumulation. After Angioplasty by an Antibody to PDGF.” (51) Int. Cl...... C12Q 1/68; C07K 1/14; Science (1992) 253:1129. CO7H 21/04: CO7H21/02 Libby et al., "Production of Platelet-Derived Growth Fac 52 U.S. Cl...... 435/6; 536/23.1; 530/413; tor-Like Mitogen by Smooth-Muscle Cells from Human 935/77; 935/78 Atheroma,” NEJM (1988) 318:1493. 58) Field of Search ...... 435/6: 935/77, Kavanaugh et al., “Transcriptional Regulation of the A and 935/78; 530/413: 536/23.1 B Chain Genes of Platelet-derived Growth Factor in Microvascular Endothelial Cells,” J. Biol. Chem. (1988) 56 References Cited 263:8470. Lindner et al., “Role of Basic Fibroblast Growth Factor in U.S. PATENT DOCUMENTS Vascular Lesion Formation." Circulation Research (1991) 4,748,156 5/1988 Aoki et al. . 68:106. 5,270,163 12/1993 Gold et al. Mann et al., "Prothrombin." Meth. Enzymol. (1981) 80:286 FOREIGN PATENT DOCUMENTS 302. WO 91/02750 of OOOO WIPO : (List continued on next page.) WO 91/1983 of OOOO WIPO : Primary Examiner-Stephanie W. Zitomer OTHER PUBLICATIONS Attorney, Agent, or Firm-Mark L. Bosse Bock et al., Nature 356:564-566 (6 Feb. 1992). 57 ABSTRACT Kadonaga et al., PNAS, USA 83: 5889-5893 (Aug. 1986). A method for identifying oligomer sequences, optionally Ullman et al. "Principles of Homogeneous -Immu comprising modified base, which specifically bind target noassay." Enzyme-Immunoassay 5:105-134 (1988). molecules such as serum proteins, kinins, eicosanoids and Van Lente et al., "Determination of Thyroxine by Enzyme extracellular proteins is described. The method is used to Immunoassay.” Enzyme-Immunoassay 6:135-153 (1988). generate aptamers that bind to serum Factor X, PDGF, FGF. Blackwell et al., “Differences and Similarities in DNA-B- ICAM, VCAM, E-selectin, thrombin, bradykinin, PGF2 and inding Preferences of MyoD and E2A Protein Complexes cell surface molecules. The technique involves complex Revealed by Selection." Science (1990) ation of the target molecule with a mixture of oligonucle 250:1104-1110. otides containing random sequences and sequences which Blackwell et al., "Sequence-Specific DNA Binding by the serve as primer for PCR under conditions wherein a com c-Myc Protein.” Science (1990) 250:1149-1152. plex is formed with the specifically binding sequences, but Tuerk and Gold, "Systematic Evolution of Ligands by not with the other members of the oligonucleotide mixture. Exponential Enrichment: RNALigands to Bacteriophage T4 The complex is then separated from uncomplexed oligo DNA .” Science (1990) 249:505-510. nucleotides and the complexed members of the oligonucle Saiki et al., "Primer-Directed Enzymatic Amplification of otide mixture are recovered from the separated complex DNA with a Thermostable DNA Polymerase." Science using the polymerase chain reaction. The recovered oligo (1988) 239:487-491. nucleotides may be sequenced, and successive rounds of Kinzler and Vogelstein. "Whole Genome PCR: Application selection using complexation, separation, amplification and to the Identification of Sequences Bound by Gene Regula recovery can be employed. The oligonucleotides can be used tory Proteins." Nucleic Acids Res. (1989) 17:3645-3653. for therapeutic and diagnostic purposes and for generating Kinzler and Vogelstein, "The GLI Gene Encodes a Nuclear secondary aptamers. Protein Which Binds Specific Sequences in the Human Genome." Mol. Cell. Biol. (1990) 10:634-642. 12 Claims, 6 Drawing Sheets 5,756,291 Page 2

OTHER PUBLICATIONS Random-Sequence Oligonucleotides: Analysis of Yeast Ellington et al., "Selection in vitro of single-stranded DNA GCN4 Protein." Mol. Cell Biol. (1989) 9(7):2944-2949. molecules that fold into specific ligand-binding structures." Chittenden et al., "The T/E1A-Binding Domain of Retino Nature (1992) 355:850-852. blastoma Can Interact Selectively with a Sequence Riordan et al., "Oligonucleotide-based therapeutics." Specific DNA-Binding Protein." Cell (Jun. 14, 1991) Nature (1992) 350:442-443. 65:1073-1082. Oliphant et al., "Defining the Consensus Sequences of E. Huynh-Dinh et al., "Modified oligonucleotide as alterna coli Promoter Elements by Random Selection." Nucl. Acids tives to the synthesis of mixed probes for the screening of Res. (1988) 16(15):7673-7683. cDNA libraries." PNAS (1985) 32:7510-7514. Oliphant et al., “Defining the Sequence Specificity of Kirk-Othmer, "Encyclopedia of Chemical Technology,"3rd DNA-Binding Proteins by Selecting Binding Sites from Ed., 6:35-54 (1979). John Wiley & Sons, NY. U.S. Patent May 26, 1998 Sheet 1 of 6 5,756,291

1541C GGTTG G - I - T G T G G T T G G T 1653 A G G T TIG G -- T G T G G G T G GIG 1715 C G G G T G G -- A TI AG G T TIGG A 1923 T G T G T G G T TACT G G T TGG G 20 27 GGGTTT G T G -- T. CTGG G T G G A 256it. T G T G T TGG -- GT TGG G T G G A 2225 T G T G T T G T G -- C C AG G T T G G A 23 12 CTTAG C G G - C A G T G G T TIG GIG 2425 TGG G T G G -- GG A G G T T G G T 2549 AGGTTTGG -- TTTGG G T G G T 26 28 AG G T T G G - T TAGGGTT G G T 2718 GGG AT GIC -- G G T G G T TGG G 2855 T G T G T TIGG - TT A T G G T T G T G T 2923 AGGTTGG T G T G G T G GC 3040AGGTTGG T G T G G GTGG G 32 (2G G T G T T G T G T G T G T G T G G A T G G T TT Consensus sequence it:T G H.EEEEEEEEEET G T TGG N3 G G T G G

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U.S. Patent May 26, 1998 Sheet 4 of 6 5,756,291

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5 HO-AGAATACTCAAGCTTGCCG-No-ACCTGAATTCGCCCTATAG-OH 3'. aptamers having little to no predetermined sequence con straints should be identified and selected for. One cannot eliminate A priori the potentially deleterious effects of 65 DNA 19-mers with the following sequences can also be used particular sequences which might arise inputative aptamers. as primers for PCR amplification of oligonucleotides recov For this reason, in the inventive methods for identifying ered from selection columns. The 3' primer sequence is 5,756,291 105 106 C. Amplification of Selected Thrombin Aptamers 5' biotin-O-CTAAGGGCGAATTCAGGTOH3' Round 1 DNA from Example 6-B was resuspended in 100 1 of HO and amplified by PCR. A 200 l PCR reaction and the 5' primer sequence is consisted of the following: 100 1 template 96-mer DNA (approximately 0.01 moles); 20110xbuffer (100 mM Tris-Cl 5' HO-AGAATACTCAAGCTTGCCG-OH3. (pH 8.3).500 mMKC1, 20 mM MgCl);321 dNTP's (5 mM It will be noted that in all cases, the duplex form of the conc total. 1.25 mM each dATP, dCTP, DGTP, and dTTP); primer binding sites contain restriction enzyme sites. 20 primer 1 (biotinylated 18-mer, 50M); 20 1 primer 2 B. Isolation of Thrombin Aptamers Using Thrombin Immo (18-mer. 50M); 6 L-P-dNTP's (approximately 60 Ci); and bilized on a Lectin Column 10 21 Taq I Polymerase (20 units). The reaction was covered A pool of aptamer DNA 96 bases in length was synthe with 2 drops NUJOL mineral oil. A control reaction was also sized as described in Example 6-A, and then PCR-amplified performed without template aptamer. to construct the initial pool. A small amount of the enzymatically-synthesized DNA was further amplified in the Initial denaturation was at 94 C. for 3 minutes, but presence of -P-dNTPs to generate labeledaptamer to subsequent denaturation after each elongation reaction permit quantitation from column fractions. 15 lasted 1 minute. Primer annealing occurred at 56 C. for 1 A thrombin column was prepared by washing 1 ml (58 minute, and elongation of primed DNA strands using the Taq nmole) agarose-bound concanavalin A ("Con-A") (Vector polymerase ran at 72 C. for 2 minutes, with 5-second Laboratories, cat, no. AL-1003) with 20 mM Tris-acetate extensions added at each additional cycle. The final elon buffer (pH 7.4) containing 1 mM MgCl2, 1 mM CaCl2, 5 gation reaction to completely fill in all strands ran for 10 mM KCl and 140 mM NaCl (the "selection buffer") (4x10 minutes at 72 C., and the reaction was then held at 4 C. ml). 1 ml of settled support was then incubated overnight at 18 rounds of Taq polymerase elongation were carried out 4C. in 10 miselection buffer containing 225 g (6.25 nmole) in order to amplify the selected aptamer DNA. After the thrombin (Sigma. Cat. no. T-6759). After shaking overnight reactions were completed, the aqueous layer was retrieved to permit thrombin binding to the Con-A beads, the mixture and any residual NUJOL oil was removed by n-butanol was briefly centrifuged and the supernatant removed. The extraction, reducing the volume to 100 L. A sample may be beads were resuspended in fresh selection buffer and trans removed from each of the aptamer and control reaction for ferred to a column which was then washed with selection quantitation and analytical PAGE. The amplified aptamer buffer (5X1 ml). A column containing 1 ml of settled beads pool (100 L) was run over a Nick column (G-50 Sephadex. had avoid volume of approximately 300 L. A control Con-A washed with 3 mLTE buffer (10 mM TrishCl(pH 7.6).0.1 column was prepared by adding 1 ml of settled support to a 30 mM EDTA)) to remove unincorporated NTP's, primers, and column followed by 5 washes of 1 ml of selection buffer. salt. 400 L of TE buffer was then added to the column and Prior to application of the aptamer DNA pool to Con-A the DNA pool was eluted from the column with an additional columns, the DNA was heated in selection buffer at 95 C. for 4001 using TE buffer. (A sample may be removed from the 3 minutes and then cooled on ice for 10 minutes. The pool, eluent for quantitation and analytical PAGE.) The eluent consisting of 100 mole DNA in 0.5 ml selection buffer, was 35 (400 L) was loaded on an avidin agarose column (Vector then prerun on the control Con-A column at room tempera Laboratories, Cat. No. A-2010) (500 L settled support, ture to remove species that bound to the control support. washed with 3x1 mL Tris/NaCl buffer (0.1M Tris, 0.1M Three additional 0.5 ml aliquots of selection buffer were NaCl, pH 7.5)). Approximately 90% of the loaded radioac added and column fractions 2, 3 and 4 (0-5 ml each) were tivity remained on the column. The column was washed with pooled and then reapplied to the column twice. The DNA in 40 Tris/NaCl buffer (4x400 l) and then the nonbiotinylated 1.5 ml selection buffer was then recovered. Approximately strand was eluted with 0.15N NaOH (3x300 l fractions). 1% of total input cpm were retained on the column. More than 45% of the radioactivity on the column eluted in The recovered DNA was then applied to a Con-A- these three fractions. These fractions (900 l) were combined thrombin column as a 0.5 ml aliquot followed by a 1.0 ml and neutralized with approximately 3.5 l of glacial acetic aliquot. Flow-through was retained and reapplied to the 45 acid. The neutralized fractions were reduced to 250 l by column twice. DNA added to the column on the final speed vacuum or butanol extraction and the nucleic acids application was left on the column for 1 hour at room were precipitated with ETOH. The resultant pellet was temperature. The column was then eluted with 0.5 ml dissolved in 1021 selection buffer. A21 sample was removed aliquots of selection buffer, 0.5 ml fractions were collected for quantitation and analytical PAGE. The resulting ampli and radioactivity was determined in each fraction. Radio 50 fied Round 1 Pool was applied to a new Con-A-thrombin activity in eluted fractions 7 through 12 were low and column as in Example 22 to obtain Round 2 aptamers. relatively constant. After recovery of fraction 12, the column D. Characterization of Round 1 Through Round 5 Thrombin was washed with 0.5 ml aliquots of 0.1M -methyl Aptamers Obtained from Selection on Lectin Columns mannoside (Sigma Cat. no. M-6882) in selection buffer to Five rounds of thrombinaptamer selection and amplification elute the bound thrombin along with thrombin-bound 55 were carried out using Con-A-thrombin columns as in aptamers. Fractions 14 and 15 showed a significant peak of Examples 6-B and 6-C. As shown in Table 5, the combined thrombin enzyme activity, as determined spectrophotometri fractions 14 and 15 contained a maximum of about 10% of cally by conversion of a chromogenic substrate (Kabi input DNA using the described conditions. Diagnostica, Cat. no. S-2238). 0.01% of the input DNA eluted in these two fractions. TABLE 5 Aptamer DNA (Round 1 DNA) was recovered from the thrombin by phenol extraction (2x0.5 ml). The aqueous % DNA eluted by % DNA bound to phase volume was reduced to about 250 l by n-butanol extraction. Aptamer DNA was precipitated on dry ice using Round -methyl-mannoside" control support 3 volumes of ethanol and 20 g of glycogen as a carrier. The 65 1. 0.01 0.7 DNA was pelleted, washed once in 70% ethanol and then 2 0.055 19 dried. 5,756.291 107 108 ml selection buffer. After application of thrombin mixture, TABLE 5-continued the filters were washed three times with 1 m selection buffer. The radioactivity retained on the filters was then % DNA eluted by % DNA bound to determined. K values for the individual clones ranged from Round -nethyl-mannoside control support 5 50 to >2000 nM. The DNA sequence of the 60-nucleotide randomly 3 5.80 2.3 generated region from 32 clones was determined in order to 4. 10.25 1.1 examine both the heterogeneity of the selected population 5 9.70 10 and to identify homologous sequences. Sequence analysis *0.1M-methyl-mannoside inselection buffer was added as fraction 13 in each 10 showed each of the 32 clones to be distinct. However, elution, and fractions 14 and 15 were retained and the DNA amplified. Due striking sequence conservation was found. The hexamer 5' to slow leeching of thrombin from the column, DNA bound to thrombin could GGTTGG 3' was found at a variable location within the also be seen in earlier fractions in rounds 3-5. random sequence in 31 of 32 clones, and five of the six After amplification, round 5 aptamer DNA was analyzed nucleotides are strictly conserved in all 32. Additionally, in for specificity in a filter binding assay. In this assay, nitro- 15 28 of the 32 clones a second hexamer 5' GGNTGG3', where cellulose filters (1 cm diameter) prebound with salmon N is usually T and never C, is observed within 2-5 nucle sperm DNA were used to bind either: (1) An unselected otides from the first hexamer. Thus, 28 clones contain the 96-mer oligonucleotide DNA pool, (2) unselected DNA with consensus sequence 5' GGNTGG(N)ZGGNTGG 3' where z thrombin (60 mole), (3) Round 5 aptamer DNA and throm is an integer from 2 to 5. The remaining 4 clones contain a bin (60 mole), (4) Round 5 aptamer DNA alone, or (5) "close variant sequence” (a sequence differing by only a Round 5 aptamer DNA and ovalbumin (60 mole). In each single base). A compilation of the homologous sequences case 3.5 mole of DNA was used and the incubation was in are shown in FIG. 1. It should be noted that DNA sequencing 200 L selection buffer at room temperature for 1 hour. The of several clones from the unselected DNA population or filters were then washed 3 times with 3.0 m of Selection from a population of aptamers selected for binding to a buffer and radioactivity was counted to determine the 25 different target revealed no homology to the thrombin amount of DNA that was retained as a thrombin complex. selected aptamers. From these data we conclude that this The results are shown in Table 6. consensus sequence contains a sequence which is respon sible either wholly or in part, for conferring thrombin affinity TABLE 6 to the aptamers. DNA % DNA Bound to Filter 3O Clotting time for the thrombin-catalyzed conversion of fibrinogen (2.0 mg/ml in selection buffer) to fibrin at 37 C. Unselected 96-mer O.08 was measured using a precision coagulation timer apparatus Unselected 96-mer + thrombin 0.06 Round 5 aptamer + thrombin 20.42 (Becton-Dickinson, Cat. nos. 64015. 64019, 64020). Throm Round 5 aptamer 0.07 bin (10 nM) incubated with fibrinogen alone clotted in 40 Round 5 aptamer + ovalbumin OOS 35 sec, thrombin incubated with fibrinogen and P1 nuclease (Boehringer-Mannheim, Indianapolis, Ind.) clotted in 39 Unselected DNA did not show significant binding to the sec, thrombin incubated with fibrinogen and aptamer clone thrombin while selected aptamer DNA bound to thrombin. #5 (200 nM) clotted in 115 sec, and thrombinincubated with Binding results show specific thrombin binding with no fibrinogen, clone #5 (200 nM) and 1 nuclease clotted in 40 detectable ovalbumin binding. sec. All incubations were carried out at 37 C. using reagents Round 5 aptamer DNA was then amplified using the prewarmed to 37 C. Aptamer DNA or, when present, P1 following 3' primer sequence: nuclease, was added to the fibrinogen solution prior to addition of thrombin. These results demonstrated that (i) thrombin activity was inhibited specifically by intact 5"HOTAAACGACCACTATAGGGATCCGAGCTCCACGTG-OH 3' 45 aptamer DNA and (ii) that inhibitory activity by aptamer did not require a period of prebinding with thrombin prior to mixing with the fibrinogen substrate. and the 5' 18-mer primer sequence shown in Example 21. Inhibition of thrombin activity was studied using a The 36-mer primer was used to generate internal BamHI consensus-related sequence 7-mer, 5' GGTTGGG 3', or a restriction sites to aid in cloning. The amplified Round 5 50 control 7-mer with the same base composition but different aptamerDNA was then cloned intopCEM32 (Promega).32 sequence (5' GGGGGTT3"). Clotting times were measured of the resulting clones were then amplified directly using the using the timer apparatus as above. The thrombin clotting following 5' primer sequence: time in this experiment was 24 sec using thrombin alone (10 nM), 26 sec with thrombin and the control sequence at 20M SOCTGCAGGTCGACGCTAGC-OH3 55 and 38 sec with thrombin plus the consensus sequence at and the 3' biotinylated 18-mer primer sequence shown in 2OM, indicating specificity for thrombin inhibition at the Example 21, and then sequenced. level of the 7-mer. Filter binding assays using aptamer DNA from 14 of the The inhibitory aptamers were active at physiological clones were used to determine the dissociation constants temperature under physiologic ion conditions and were able (K) for thrombin as follows: Thrombin concentrations 60 to bind to thrombin in the presence of the fibrinogen between 10 m and 1 nM were incubated atroom temperature substrate, a key requirement for therapeutic efficacy. in selection buffer for 5 minutes in the presence of 0.08 mole of radiolabeled 96-mer derived from cloned Round 5 Example 7 aptamer DNA. After incubation, the thrombin and aptamer mixture was applied to nitrocellulose filters (0.2 micron, 2.4 65 Modified Thrombin Aptamers cm diameter) that were pretreated with salmon sperm DNA Modified forms of the single-stranded, thrombin consen (1 mg/ml DNA in selection buffer) and washed twice with 1 sus sequence-containing deoxynucleotide 15-mer described 5,756.291 109 110 in Example 7.5' GGTTGGTGTGGTTGG 3', and a closely 2'-deoxycytidine are synthesized using methods described related 17-mer, were synthesized using conventional tech above. Each compound is converted to the triphosphate form niques. These aptamers for the most part contain the iden and tested in the PCR assay described in Example 1 using an tical nucleotide sequences, bases, sugars and phosphodiester appropriate mixture of three normal deoxytriphosphates or linkages as conventional nucleic acids, but substitute one or more modified linking groups (thioate or MEA). or modified ribotriphosphates along with a single modified base analog. bases (uracil or 5-(1-pentynyl-2'-deoxy)uracil). The aptam This procedure may also be performed with Nposition ers containing 5-(1-pentynyl)-2'-deoxyuridine were gener alkylated analogs of adenosine and 2'-deoxyadenosine, and ated by replacing thymidine in the parentaptamers. Throm bin aptamers containing 5-(1-pentynyl)-2'-deoxyuridine the 7-position alkylated analogs of deazaguanosine, deaza were also obtained by selection as described in Examples 13 10 2'- deoxyquanosine, deazaadenosine and deaza-2'- and 14 below. deoxyadenosine, synthesized using methods described in the Independent verification of the K, for the nonmodified specification. 15-mer was made by determining the extent of thrombin inhibition with varying DNA concentration. The data Example 10 revealed 50% inhibition of thrombin activity at approxi 15 mately the same concentration as the derived K, strongly suggesting that each bound thrombin was largely, if not Thrombin Aptamer Containing Substitute completely, inhibited, and that binding occurred with a 1:1 Internucleotide Linkages stoichiometry. Modified forms of the 15-mer thrombinaptamer, 5' GGT TABLE 7 TGGTGTGGTTGG 3' containing one or two formacetal internucleotide linkages (O-CH-O) in place of the phos Compound K (nM) phodiester linkage (O-PO(O)-O) were synthesized and GGTTGGTGTGGTTGG 20 assayed for thrombin inhibition as described above. The GGTTGGTGTGGTTGGGFT 35 25 GGTTGGTGTGGTTFGFG 40 H-phosphonate dimer synthon was synthesized as described GGTTG-G-TAGTGGTTGG 280 in Matteucci, M. D. Tet. Lett. (1990) 31:2385-2387. The GGTTGG(dUG(dUGGTTGG 15 formacetal dimer, 5'TO-CH-O-T3'. was then used in solid GG(dU)TGGTGTGG(dU)TGG 80 GGTTGGTGTGGTUGG 20 phase synthesis of aptamer DNA. Control unmodified 30 aptamer DNA was used as a positive control. *indicates a thioate (i.e., P(O)S) linkage #indicates a MEA linkage The results that were obtained are shown in Table 8. U'indicates 5-(1-pentynyl)uracil Example 8 TABLE 8 35 Incorporation of 5-(1-pentynyl)-2'-deoxyuridine clot time (sec) Into Aptamer Candidate DNA Compound 100 nM. 20 nM 0 nM 5-(1-pentynyl)-2'-deoxyuridine was synthesized and con GGTTGGTGTGGTTGG 105 51 m verted to the triphosphate as described in Otvos, L., et al., GGTTGGTGTGGTTGG 117 48 m Nucleic Acids Res (1987) 1763-1777. The pentynyl com GGTTGGTGTGGTGG 84 60 pound was obtained by reacting 5-iodo-2'-deoxyuridine with GGTTGGGGGGG 125 49 1-pentyne in the presence of palladium catalyst. 5-(1- NO DNA CONTROL - m 25 pentynyl)-2'-deoxyuridine triphosphate was then used as a replacement for thymidine triphosphate in the standard PCR reaction. 45 indicates a formacetal linkage A pool of 96-mer single-stranded DNA was synthesized, each strand consisting of specific 18-mer PCR primer Example 11 sequences at both the 5' and 3' ends and a random 60-mer sequence in the center of the oligomer. Details of synthesis Thrombin Aptamer Containing Abasic Nucleotide of the pool of single-stranded DNA is disclosed in Examples 50 Residues 1-6 above. PCR conditions were the same as those described above, with the following changes. dATP, DGTP and dCTP Modified forms of the 15-mer thrombinaptamer, 5' GGT. were all used at a concentration of 200M. The optimal TGGTGTGGTTGG 3' containing one abasic residue at each concentration for synthesis of full-length 96-mer DNA via position in the aptamer were synthesized and assayed for PCR using 5-(1-pentynyl)-2'-deoxyuridine was 800M. Gen 55 eration of PCR-amplified fragments demonstrated that the thrombin inhibition as described above. The abasic residue, Taq polymerase both read and incorporated the base as a 1,4-anhydro-2-deoxy-D-ribitol was prepared as described in thymidine analog. Thus, the analog acted as both substrate Eritja, R. et al. Nucleosides and Nucleotides (1987) and template for the polymerase. Amplification was detected 6:803-814. The N,N-diisopropylamino cyanoethylphos by the presence of a 96-mer band on an EtBr-stained phoramidite synthon was prepared by standard methods as polyacrylamide gel. described in Caruthers, M. H. Accounts Chem. Res. (1991) 24:278-284, and the derivatized CGP support was prepared Example 9 by the procedures described in Dahma, M. J., et al, Nucleic Incorporation of Other Base Analogs Into Acids Res. (1990) 18:3813. The abasic residue was singly Candidate Aptamer DNA 65. substituted into each of the 15 positions of the 15-mer. Ethyl, propyl and butyl derivatives at the 5-position of Control unmodified aptamer DNA was used as a positive uridine, deoxyuridine, and at the N' position of cytidine and control. The results that were obtained are shown in Table 9. 5,756.291 111 112 A pool of 60-mer single-stranded DNA was synthesized, TABLE 9 each strand consisting of specific 18-mer PCR primer sequences at both the 5' and 3' ends and a random 20-mer clot time (see) sequence in the center of the oligomer. Details of synthesis Compound OOM of the pool of single-stranded DNA is disclosed in Example 1. GGTTGGGGGGX 27 GGTTGGTGTGGTTXG 27 Because of the poor substrate activity of pentynyl dOTP GGTTGGGTGGXGG 27 when used with TAO polymerase, VENT-thermostable GGTTGGTGTGGXTGG 56 polymerase, (New England Biolabs, Cat. No. 254) was GGTTGGTGTGXTGG 27 10 employed. Amplification was performed as per the manu GGTTGGTGTXGTGG 29 GGTTGGTGXGGTTGG 43 facturers instructions. Pentynyl dITP was included in the GGTTGGXGGTGG 5 reaction as a substitute for dTTP. The single-stranded GGTTGGXGTGGGG 6 60-mer was isolated by a modification of standard proce GGTTGXTGGGGG 27 dures. The 200 L PCR amplification reaction was divided GGTTXGTGGGTTGG 2. 15 GGTXGGTGTGGTGG 27 into two samples which were applied to two NICK8 col GGXTGGTGTGGTGG 62 umns equilibrated (5 mL) as described. The eluent was GXTTGGTGTGGGG 27 collected, pooled and applied to avidin-agarose as described. XGTTGGGTGGGG 28 This column was washed with buffer followed by elution of GGTTGGTGTGGTTGG 136 single-stranded 60-mer DNA with 0.15N NaOH, pooled and NO DNACONTROL neutralized with glacial acetic acid. Single-stranded 60-mer X- indicates an abasic residue DNA was desalted on a NAP5 column equilibrated in 20 mM Tris OAc (pH 7.4). 10xselection buffer salts were added Example 12 to the sample, heated to 95 C. for 3 minutes, and transferred to wet ice for 10 minutes. Thrombin Aptamers Containing -5-(1-Propynyl)-2'- 25 deoxyuridine Nucleotide Residues Example 14 Modification of the 15-mer thrombin aptamer, 5' GGT. Isolation of Thrombin Aptamers Using DNA TGGTGTGGTTGG 3' to contain 5-(1-propynyl)-2'- Containing 5-(1-Pentynyl)-2'-deoxyuridine deoxyuridine nucleotide analogs at the indicated positions in the aptamer was effected by the synthesis of these aptamers. 30 The pool of aptamer DNA 60 bases in length was used They were assayed for thrombin inhibition as described essentially as described in Example 13. The aptamer pool above. The aptamer and the H-phosphonate were prepared sequence was as described in DeClercq, E. et al., J. Med.Chem. (1983) 5TAGAATACICAAGCITCGACG-N-AGTTTGGAICCCCGGGTAC 3', 26:661-666; Froehler, B. C., et al, Nucleosides and Nucle 35 otides (1987) 6:287-291; and Froehler, B. C. etal, Tet, Lett. while the 5' primer sequence was (1986) 27:469. This analog residue was substituted at the indicated positions and the aptamer assayed for inhibition of 5'TAGAAACTCAAGCCGACG 3' thrombin. The results that were obtained are shown in Table 10. and the 3' biotin-linked primer was

TABLE 10 5 GTACCCGGGGACCAAACT 3. Thrombin immobilized on a Con-Alectin column served as clot time (see) the target as described. Compound 100 nM OM 45 After five rounds of selection, aptamer DNA was recov GGTTGGTGTGGGG 4. ered and amplified using thymidine triphosphate (dTTP) in GGTTGGGTGG2GG 29 place of 5-(1-pentynyl)-2'-deoxyuridine in order to facilitate GGTTGGTG2GGTTGG 20 subsequent cloning and replication of aptamer DNA in E. GGTTGGZGTGGGG 118 coli. At this stage, the presence of a thymidine nucleotide at GGZGGTGGGTTGG 18 50 GGZGGGGGTTGG 138 a given location in an aptamer corresponded to the location GGTTGGGTGGTGG 125 of a 5-(1-pentynyl)-2'-deoxyuridine nucleotide in each origi NODNACONTROL nal round five aptamer. Thus, dTTP served to mark the location of 5-(1-pentynyl)-2'-deoxyuridine residues in the Z - indicates a 5-propynyl-2'-deoxyuridine residue original selected DNA pools. 55 The round five amplified DNA containing dTP was Example 13 digested with BamHI and HindIII and cloned into the corresponding sites of pCEM 3Z (Promega Biotech) and Incorporation of 5-(1-pentynyl)-2'-deoxyuridine transformed into E. coli. DNA from 21 clones was analyzed Into Aptamer Candidate DNA by dideoxy sequencing. Three of the clones contained 5-(1-pentynyl)-2'-deoxyuridine was synthesized and con aptamer sequences that were identical. Only one of the 21 verted to the triphosphate as described in Otvos, L., et al., clones contained a sequence that closely resembled the Nucleic Acids Res (1987) 1763-1777. The pentynyl com original 5' GGTTGG 3' binding motif obtained using thym pound was obtained by reacting 5-iodo-2'-deoxyuridine with ine in the selection protocol. 1-pentyne in the presence of a palladium catalyst, 5-(1- One of these two clones (#17) and the original unselected pentynyl)-2'-deoxyuridine triphosphate was then used as a 65 pool was analyzed for thrombin binding by nitrocellulose replacement for thymidine triphosphate in the standard PCR filter assay described above using DNA labeled with P to reaction. permit analysis of thrombin binding characteristics. The 5,756.291 113 114 labeled DNA was synthesized by PCR and contained 5-(1- third pass the peak radioactive element was then applied to pentynyl)-2'-deoxyuridine in order to retain the original a 1 mL ConA/thrombin column (charged with 3 nmoles of selected DNA structures. The DNA was incubated with thrombin). Radioactive single-stranded 98-mer was applied thrombin at various concentrations between 10 nM and 10M three times to this matrix. At the third application, the to obtain the K values for thrombin binding. The K of the column was stoppered and allowed to stand for 1 hr. The unselected pool was >10 m while the K of clone 17 was column was then washed with selection buffer and 0.5 mL 400 nM. aliquotfractions collected. A total wash volume of 6 mL was Radiolabeled clone 17 DNA was synthesized using thy employed. At this time. 0.1 m -methyl-mannoside in selec midine in place of 5-(1-pentynyl)-2'-deoxyuridine and the tion buffer was then added, followed by a 4 mL total volume resulting DNA had a K of >10M, demonstrating that the O wash. Thrombin enzymatic activity was detected via chro 5-(1-pentynyl)-2'-deoxyuracil heterocycle could not be mogenic substrate monitored by absorbance at 405 nm. Peak replaced by thymine in the selected aptamer without loss of thrombin fractions were pooled, extracted with phenol, and binding affinity. the volume reduced by nBuOH extraction. 20 g glycogen Representative sequences that were obtained are as foll was added, the single-stranded 98-mer precipitated via etha lows. 15 nol addition and pelleted via centrifugation. The pelleted DNA was resuspended in water and used as a template for 5'TAGTATGTATATGTGTAG 3' PCR amplification. This protocol was repeated to obtain a 5'ATAGAGTATAATGCTGTCT3' pool of DNA that resulted from 5 rounds of selection on 5' GTATATAGTATAGTATTGGC 3' thrombin columns. 5'AGGATATATGAATGATTCGG 3' 5'TACTATCATGTATATTACCC 3' Double-stranded DNA was digested with EcoRI and 5' CATTAAACGCGAGCTTTTTG 3' 5' CTCCCAAATGCCCTAGCCG 3' HinDIII and cloned into pGEM32. Aptamers were then 5' GACGCACCGACCCCGT 3' transformed into E. coli and analyzed by dideoxy sequenc 5' CACCAAACGCATTGCATTCC 3' ing. Round five aptamer pool DNA bound to thrombin with 5 GTACATTCAGGCTGCCTGCC 3' a K of approximately 300 nM. 5'TACCATCCCGTGGACGTAAC 3' 25 5' GACTAAACGCATTGTGCCCC 3' 5'AACGAAGGGCACGCCGGCTG 3' Example 16 5'ACGGAGGTCTGGCGGACA3' Demonstration of Aptamer Specificity for Binding to and Inhibition of Thrombin Example 15 30 The specificity of aptamer binding was demonstrated Isolation of Thrombin Aptamers Using using 'P radiolabeled DNA and a series of proteins. To DNA Containing 5-Methyl-2'-deoxycytidine 5-methyl-2'- determine the binding specificity of the thrombin aptamer, deoxycytidine triphosphate was obtained commercially 96-mer clone #29, having the partial sequence 5' CGGG (Pharmacia, Cat. No. 27-4225-01) and used to synthesize 35 GAGAGGTT GGTGTGGTTGG CAATGGCTA DNA containing random sequences 60 bases in length GAGTAGTGAC GTTTTCGCGGTGAGGTCC 3' was flanked by primers 19 bases in length. The pool of aptamer used. The consensus sequence is shown underlined. In DNA 98 bases in length was used essentially as described in addition, a 21-mer aptamer, 5' GGTTGGGCTGGTTGGGT. Example 6. Thrombin immobilized on a Con-A lectin col TGGG 3' was tested for inhibition of another fibrinogen umn served as the target as described. cleaving enzyme ancrod, which was obtained commercially Briefly, a 200 L PCR reaction was set up using: 10 mM (Sigma, Cat. No. A-5042). The 21-mer had a of K for Tris-HCl, pH 8.3 at 25 C. 1.5 mM MgCl, 50 mMNaCl and thrombin of about 100 nM and its K was about 350 nM. 200 m of each of dATP, DGTP, dTTP and 5-methyl-2'- Clone #29 had a K of about 200 nM for thrombin. deoxycytidine triphosphate. 20 Ci each of -'P-dATP and The aptamer was shown to specifically bind to thrombin dGTP were added to label the DNA. 1 nmole of 5" and 3' 45 by a filter binding assay. Briefly, radiolabeled aptamer DNA primer were added followed by addition of 0.2 mole of at about a concentration of about 1 nM was incubated with 98-mer template pool DNA. Amplification was initiated by the indicated protein for several minutes at room addition of 21 (10 U) of Taq polymerase followed by sealing temperature, followed by filtration of the aptamer-protein of the reaction with a mineral oil overlay. About 16 cycles mixture through a nitrocellulose filter. The filter was washed of amplification were performed followed by a 10 minute 50 with 3 mL of selection buffer and then radioactivity bound final extension to complete all duplex synthesis. to the filters was determined as a % of input radioactivity. Amplified DNA was recovered (100 L aqueous phase), Results obtained are shown in Table 11. Binding data is n-butanol extracted (650 L) and applied to a Nick column shown for both unselected 96-mer DNA and for two separate prewashed with 5 mL of buffer containing 100 mM TrishCl experiments with clone #2996-mer. All proteins were tested pH 7.5 and 100 mM. NaCl. Eluted DNA was applied to a 0.5 55 at about 1M concentration except human serum albumin mLavidin-agarose column prewashed in the same buffer and which was used at 100M. The results that were obtained washed until DNA loss from the column was <1000 cpm. demonstrated that the 96-mer specifically bound to thrombin Single stranded DNA was eluted from the avidin column by and had little affinity for most of the other proteins tested. washing with 0.15N NaCl and the eluate was neutralized to pH 7.0 using glacial acetic acid. The 98-mer DNA was TABLE 11 exchanged into selection buffer on a second Nick column and, after heat denaturation for 3 min at 95 C. followed by Protein Input CPM Bound CPN 96 Bound cooling on ice for 10 min, used in aptamer selection on thrombin lectin columns. 1 mL ConA/thrombin columns Unselected DNA were equilibrated in selection buffer prior to addition of 65 Control 75573 230 O single-stranded DNA. The single-stranded DNA was recir Thrombin 74706 6732 9.O culated for three complete passes. Upon completion of the 5,756,291 115 116 prolongation occurring at the higher dose concentration. TABLE 11-continued Little or no activity was observed at 20 minutes. Thus, the 15-mer in blood withdrawn from rats 5 minutes post Protein Input CPM Bound CPN Bound injection was able to inhibit exogenously added human Prothrombin 75366 183 a).5 thrombin. A separate APTT test at the 5 minute time point Albumin 16560 1851 2.0 showed that the 15-mer also inhibited rat blood coagulation. Chymotrypsin 7.5566 225 a).5 presumably by inhibiting rat thrombin to a significant Trypsin 739.93 306 KO.S Kalikrein 76066 122 O.5 degree. The half-life of the 15-mer in rats appears to be Plasmin 7453 3994 50 about 2 minutes or less. Clone 29 DNA 10 Example 18 Control 8,280 126 O Throbin 8753 4816O 59.0 Thrombin Aptamer Primate Studies Prothrombin 81580 8849 110 Albumin 85873 778 2.0 Two thrombinaptamers were administered to adult male Chymotrypsin 82953 2O7 CO.5 15 cynomologous monkeys. Unsubstituted 15-mer DNA with Trypsin 7563 318 a).5 the sequence 5' GGTTGGTGTGGTTGG 3' and an analog, Kallikrein 84013 143 O.5 5' GGTTGGTGTGGTTG*G 3', containing thioate inter Plasmin 82633 12323 15.0 TPA 81960 192 g).5 nucleotide linkages at the indicated positions were used. Cone 29 DNA Aptamer was delivered as an intravenous bolus or infusion and then blood samples were withdrawn at various times Control 81886 91. O after delivery of the bolus or during and after infusion. The Thrombin 82.940 48796 59.0 Prothrombin 9760 8719 9.5 catheter was heparinized after the 10 minute timepoint. The Albumin 92473 234 <0.5 animals were not systematically heparinized. Chymotrypsin 97O60 186 kO.5 Thrombin inhibition was measured by a prothrombin time Trypsin 97846 429 aO.5 Kallikrein 95053 1275 O.5 25 test (PT) using a commercially available kit, reagents and Plasmin 66565 9704 S.O protocol (Sigma Diagnostics. St. Louis, catalog Nos. T0263 PA 98.66 644 O.S and 870-3). Inhibition of thrombin was indicated by an increased clot time compared to the control in the PT test, Clottimes were obtained by withdrawing a sample of blood, The thrombin 21-mer ancrod assay was conducted as 30 spinning out red cells and using the plasma in the PT test. follows. Ancrod was suspended in sterile water at a concen Control thrombin PT clot time values were obtained several tration of 44 U/mL. 10 Lancrod solution was added to 95 L minutes prior to administration of aptamer, Briefly, the PT of selection buffer prewarmed to 37 C. 100L of this mixture assay was conducted using 0.1 mL of monkey plasma was transferred to the coagulation cup of the fibrometer prewarmed to 37 C. and 0.2 mL of a 1:1 mixture of described above, followed by addition of 200L offibrinogen 35 thromboplastin (used according to manufacturers and 20 L of 21-mer DNA (both prewarmed to 37 C). TE instructions) and CaCl (25 mM), also prewarmed to 37 C. buffer pH 7.0 was used as a control lacking DNA. The Thrombin clot times were measured with a fibrometer as control clot time was 25 seconds while the clot time in the described above. presence of 500 nM 21-mer was 24 seconds and was 26 The animals were at least two years old and varied in seconds in the presence of 33M 21-mer. This result dem weight from 4 to 6 kg. Doses of aptamer were adjusted for onstrated the specificity on inhibition of fibrinogen cleavage body weight. AptamerDNA was dissolved in sterile 20 mM was limited to thrombin; ancrod was not affected. phosphate buffer (pH 7.4) at a concentration of 31.8 to 33.2 Example 17 mg/mL and diluted in sterile physiological saline prior to delivery. Bolus injections were administered to give a final Thrombin Aptamer Pharmacokinetic Studies 45 concentration of 22.5 mg/Kg (1 animal) of the diester A 15-mer single-stranded deoxynucleotide, 5' GGTTG aptamer or 11.25 mg/Kg (1 animal) of the diester aptamer. GTGTGGTTGG3', identified as a consensus sequence from Infusions were administered over a 1 hour period to three 30 thrombin aptamer clones as described in Example 6 groups of animals: (i) 0.5 mg/kg/min of diester 15-mer (4 above, was used. Young adult rats of mixed gender and animals), (ii) 0.1 mg/kg/min of diester 15-mer (2 animals) strain were used. The animals were anaesthetized and a 50 and (iii) 0.5 mg/kg/min of thioate analog 15-mer (2 diester of the 15-mer was injected through a catheter in 200 animals). lvolumes (in 20 mM phosphate buffer, pH 7.4, 0.15MNaCl) PT assay results from the bolus injections showed throm at two concentrations, so that the final concentration of bin inhibition times of 7.8, 3.3 and 1.35 times control at 2.5, 15-mer in the blood was about 0.5 and 5.0M respectively, 5.0 and 10.0 min respectively after delivery of the aptamer although the exact concentration depends on the volume of 55 for the high dose animal. Inhibition times of 5.6, 2.2 and 1.2 distribution (which is unknown for this oligonucleotide). times control were obtained from the low dose animal at the These values are 10 to 100 times greater than the human in same time points. vitro K value. No heparin was used for catheterization. FIG. 2 shows a plot of the PT times from the 4 animals At 0, 5, 20 and 60 minutes, blood was withdrawn from the that received the high dose diester infusion compared to animals (approx. 500 l aliquots), transferred into tubes pretreatment control values. The data points show the PT containing 0.1 volume citrate buffer, and centrifuged. Rat clot time as an average value obtained from the 4 animals in plasma was removed and tested in a thrombin clotting-time the group. The arrows indicate time points at the beginning assay. Six animals were used at each concentration, and and end of the infusion period. Thrombin inhibition peaked three animals were injected with the control carrier solution at about 10 to 20 min after the infusion was initiated and containing no 15-mer. 65 remained level until the infusion period was terminated. A prolonged clotting time was observed at the 5 minute Inhibitory activity decreased rapidly after the infusion of time point at both concentrations, with the most significant aptamer terminated. 5,756.291 117 118 High dose diester and high dose thioate animals showed thrombin (77 sec G. 200 nM verses 28.9 sec for the control comparable inhibition of thrombin-mediated clotting, with in the absence of DNA). However, it was also observed that the high dose thioate giving a sustained clot time of 2.5 to clotting was not dependent on the methyl group of 5-methyl 2.7 times the control value during the course of the infusion. dC. The low dose diester compound gave a clottime of 1.4 to 1.5 In parallel, the sixth round pool was cloned and times the control value. These results demonstrated the sequenced. All clones characterized were found to contain efficacy of the native and thioate analog aptamers in pri the consensus sequence (i.e. GGNTGGNNNGGTNGG). lateS. 5-methyl deoxycytidine triphosphate was from Pharmacia and the corresponding suitably protected phosphoramidite Example 19 10 from Milligen. 5-pentynyl deoxyuridine (5' DMT or PONa) was synthesized via Pd/Cu-mediated coupling of Inhibition of Extracorporeal Blood Clotting By the corresponding iodoalkyne and 5-iodo-deoxyuridine Thrombin Aptamer (Hobbs, F. W., (1989) J. Org. Chem. 54:3420-3422). Anticoagulation of a hemodialysis filter was demon 5-pentynyl deoxyuridine triphosphate (-P(OCH2CHCN) strated using the 15-mer 5' GGTTGGTGTGGTTGG 3' 15 (N(CH(CH))) was then prepared via the method of thrombin aptamer with human blood. A bolus of 15-mer Kohler et al. (1980) Helv. Chim. Acta 63:2488-2492, and DNA was delivered to human blood at 37 C. to give an the protected phosphoramidite prepared via the method of aptamer concentration of 10M. The blood was contained in Koster (1984) Nuc. Acids Res. 12:4539-4557. an extracorporeal hemodialysis circuit (Travenol, Model No. To determine the effect of introducing a larger hydropho CA-90). Pressure proximal to the hemodialysis filter was 20 bic element into the random oligomer, a random pool monitored to determine the time after administration of containing 5-pentynyl dU was prepared. A second oligomer aptamer that coagulation occurred. Blood coagulation was pool was chemically synthesized containing 20 random marked by a pressure increase from about 50 mm Hg bases flanked by two constant regions for use in PCR. This observed with uncoagulated blood (blood flow rate 200 template was then amplified in the presence of 5-pentynyl mL/min) to pressure of at least 400 mm Hg. 25 dUTP to construct the initial pool completely substituted Using citrated whole blood (recalcified at time zero), with 5-pentynyl dO in place of thymidine. The selection coagulation occurred at about 9 minutes after fresh blood procedure was then repeated in an identical format to that was placed in the hemodialysis unit and circulation was described above. After six rounds of selection and begun. (In a repeat of this control experiment, coagulation amplification, a second modified base aptamer pool towards occurred at 11 minutes.) A heparin control (1 U/mL) gave 30 thrombin was isolated. sustained anticoagulation until the experiment was termi Of the twenty-seven sequences analyzed, only one fit the nated at 80 minutes after start of circulation in the unit. GT-rich pattern highlighted above. Three sequences, repre Blood coagulation occurred at 51 minutes in one trial with senting different levels of 5-pentynyl dU substitution were the 15-mer. In a second trial, coagulation did not occur chosen for further study (clone 3, clone 7 & clone 24). In during the 80 minute course of the experiment, 35 addition, and more importantly, clone 24 represented a sequence observed in three distinct clones. Dissociation Example 20 constants of these clones were determined to be 800. 1000 and 400 nM, respectively. Using an electrophoretic mobility Modified Thrombin Aptamers shift assay (EMSA) the protein specificity of this pool was Two distinct hydrophobic additions, 5-methyl-2'- analyzed (Chodosh, L. in Current Protocols in Molecular deoxycytidine (5-methyl dO) and 5-pentynyl-2'- Biology (eds Ausubel, F. M. et al.) 12.2.1-12.21.0 (Wiley deoxyuridine (5-pentynyl dJ) were used to capture a novel Interscience, New York 1987)). interaction in thrombin-DNA interaction unavailable with A radiolabelled clone 24 oligomer was prepared, incu natural nucleotides. Using chemical synthesis a 98-base 45 bated with various concentrations of thrombin and then DNA oligomer containing a random region of 60 bases assayed via native polyacrylamide gel electrophoresis; a flanked by defined sequences for use in PCR amplification thrombin dependent band-shift was observed. was prepared. This initial oligomer pool was subjected to This radiolabelled oligonucleotide was incubated with a large scale PCR amplification in the presence of 5-methyl series of related proteins: trypsin, chymotrypsin, albumin, dCTP to prepare an initial 98-base pool containing complete 50 Factor X. prothrombin, -thrombin and thrombin. A protein substitution of 5-methyl dC for dC. This pool was then used dependent band shift was observed only in the case of in a multi-round selection protocol towards human thrombin thrombin. An electrophoretic mobility shift assay (EMSA) immobilized on a concavalin A-agarose column (Con was employed to assay protein/ssDNA aptamer interaction A-agarose). as follows: Lane 1, no protein, lane 2, thrombin; lane 3, After the sixth cycle of this process, forty percent of the 55 prothrombin; lane 4.-thrombin; lane 5, human albumin; lane input DNA was retained on the thrombin containing column 6, Factor X; lane 7, chymotrypsin; lane 8, trypsin. and was specifically eluted with -methyl mannoside. This 5'-'P-labelled pentynyl dU clone 24 aptamer was pre dissociation constant of the DNA-protein complex was then pared using-P-ATP (Amersham) and T4 polynucleotide determined via nitrocellulose filter binding and the Kd of the kinase (U.S. Biochemical) using standard procedures pool was estimated to be 200 nM compared to the unselected (Sambrook et al. in Molecular Cloning, A Laboratory 5-methyl dC pool of 10M. Manual, 2nd Ed. 10.59-10.61). The Paptamer was heated In a previous example, it was observed that aptamer to 95 C. in selection buffer (Bock (1992) Nature thrombin complex formation results in a loss of enzymatic 355:564-566) for 5 minutes and cooled to room tempera activity when assayed with a purified system. Large scale ture. This DNA was then added to the respective protein PCR amplification isolated the single strand 98 base 65 equilibrated at 4C. (final concentration-500 nM in selection oligomer, and analyzed the enzyme inhibition properties of buffer). This was allowed to stand on ice for 90 minutes. The this pool using this purified system. This pool inhibited sample was then brought to 10% glycerol and electrophore 5,756.291 119 120 sised at 4 C. using 20 mM. NaCl/50 mM HEPES pH 7.1 in property resides solely in the random region of selected a 4% (39:1) native acrylamide gel. Human thrombin, clones. Oligonucleotides of the 20 base variable region of prothrombin, -thrombin and Factor X were from Haemoto two clones (3 & 24) were then prepared via chemical logics Technologies Inc.; human albumin, chymotrypsin and synthesis. trypsin were from Sigma. These two 20 base oligomers were then analyzed for From these results it was concluded that this pool con clotting inhibition activity using purified human thrombin tained high affinity aptamers towards thrombin, and that the (Haemetologic Technologies Inc.) and human fibrinogen modified pyrimidine was required for the binding pre-equilibrated at 37 C. was added. The final concentration interaction, in distinct contrast to results obtained using of thrombin and fibrinogen in the mixture was 13 nM and 5-methyl dC. Furthermore, the results in the EMSA with O 5.9M respectively. Oligonucleotides concentrations were as -thrombin, a modified form of thrombin where the anion listed above and were attor greater than their respective Kd binding exosite has been proteolytically cleaved (Bing et al. as determined via nitrocellulose filter binding. Clotting (1977) J. Biol. Chem. 252:8027-8034), implicated the times were measured using an automated fibrometer. Results involvement of this exosite as at least one component of the of this assay is set forth in Table 12 below. binding site for this class of aptamers. 5 Nitrocellulose filter binding with purified human throm TABLE 12 bin was performed to determine the Kd of the initial Clotting Inhibition by Modified Base Containing 5-pentynyl dU random pool and the sixth round pentynyl dU Oligonucleotides aptamers. The dissociation constant of the initial pool was greater than 10M while the selected pool was 400 nM. In 20 Concentration Time (sec) addition, it was observed that the sixth round selected pool (1) Clone 24 (1M) 24 required 5-pentynyl du; no significant binding was observed GXAXAXAGXAXAGXAXXGGC when the pool was prepared with thymidine (Kd>10M). The 20 nucleotide variable region final 5-pentynyl dO pool was then cloned and a number of (2) Clone 24 (1M) 24 GCXGXAGAXAXXAGXAXAGX clones sequenced. 25 20 nucleotide variable region FIG. 3 illustrates the sequences obtained from the round scrambled 6 with 5-pentyny dily pool as follows: Sixth round single (3) GGTTGGTGTGGTTGG (20 nM) 43 (4) Clone 24-60 nucleotide (4M) O45 stranded DNA was amplified, digested and cloned into fragment with pentynyl dJ pGEM32 using HindIII and EcoRI. Twenty-seven clones (5) Clone 3-60 nucleotide (4M) 37.5 were sequenced via dideoxy methodology, Aptamer selec 30 fragment tion was performed as in (Bock 1992, supra) including the (6) Clone 24/substituted-60 (4M) 25. use of negative selection with Concavalin A?agarose (Con nucleotide fragment A?agarose). A general protocol for the selection method is as (7) Control 24.3 follows: a 60 nucleotide oligomer of the sequence X = pentynyl du 5'-TAGA ATA CT CAA G CTT CGA CG (N) 35 AGTTTGGATCCCCGGGTAC-3' was prepared via auto All three molecules were inactive. The entire 60 base mated synthesis using standard phosphoramidite chemistry. sequences of clones 3 & 24 were then prepared containing After deprotection in NHOH, followed by desalting using the random region flanked by the two constant regions and a NAP10 column (Pharmacia), the DNA was amplified incorporated 5-pentynyl dJ in the 3' constant region and T using PCR in a 200 L reaction containing: 10M primer in the 5' constant region, analogous to the PCR product. In 5'-TAGAATACTCAAGCTTCGACG-3"; 10M 5'-biotin addition, a clone 24 analog was also prepared replacing all GTACCCGGGGATCCAAACT-3'; 25 OM dGTP, 250M 5-pentynyl dU residues with thymidine. Each was then DATP 250M dCTP and 250M pentynyl dUTP; 10 mMKCl, analyzed for its ability to inhibit thrombin (Table 12 above). 10 mM (NH4)SO, 2 mM MgSO, 0.1% TritonX-100 in 20 Clotting inhibition was observed with full length clone 24, mM Tris pH 8.8 in the presence of 60 Ci-'P-dNTPs (dCTP. 45 and furthermore this activity was dependent on the presence dATP & dGTP). The reaction was brought to 100 C. for 3 of 5-pentynyl du (table 12 entry 6). minutes and 10 U of Vent Polymerase (New England From these results it was concluded that one or both of the Biolabs) was added. This was cycled 18 times through 100 constant regions of the oligomer play a significant role for C., 1 min.; 56 C. 1 min.; 75 C., 2 min. using an extension thrombin inhibition. In addition, it was independently of 5 sec/cycle at this temperature. The replacement of 50 verified, via this coagulation assay, that the interaction by thymidine by 5-pentynyl dJ in this and in all subsequent this aptamer was dependent on the 5-pentynyl du hydro amplifications results in DNA containing solely this modi phobic functionality as judged by the clone 24 thymidine fied pyrimidine in the locations originally containing thy substitution result. Sequence analysis has not established a midine in the synthetic oligonucleotide. The resulting 60 nt consensus oligonucleotide which is responsible for the bio ds DNA product was applied to a Nick column (Pharmacia) 55 logical activity. Instead, the data points to the involvement equilibrated in 100 mM NaCl/100 mM Tris pH 7.5 and of a conserved secondary or tertiary structure dependent on eluted following the manufacturer's instructions. Single 5-pentynyl dU for its formation and interaction with throm stranded 60 nt DNA was then isolated via the use of avidin bin. agarose; single stranded 60 nt DNA was eluted from the The above results demonstrates that modified nucleotides matrix using 0.25N NaOH, neutralized with glacial acetic can dramatically change the sequence of the aptamers iso acid to pH 6 and the buffer exchanged via Nick column lated through in vitro selection, and in studies with equilibrated in 10 mM Tris-acetate pH 7.4. The eluent was 5-pentynyl du the observed biochemical activity was depen then brought to 20 mM Tris-acetate containing 140 mM dent on the modified nucleotide. The inclusion of modified NaCl, 5 mM KC, 1 mM MgCl, and 1 mM CaCl. Selection nucleotides therefore clearly broader the repertoire of the was performed as described in Bocket al. (1992) supra. 65 selection process. A major functionality change. Dramatic A correlation between protein-DNA binding and the inhi changes such as 5-pentynyl du were able to alter the bition of enzymatic activity was observed. Furthermore, this outcome of the process, whether this is related to a dominant 5,756.291 121 122 A. Synthesis of Oligonucleotide Pool structural feature of the anion binding site on thrombin is not DNA oligonucleotides containing a randomized sequence known at present. region were synthesized using standard solid phase tech Example 21 niques and phosphoramidite chemistry (oligonucleotide Synthesis, Gait, M. i., ed. (IRL Press), 1984; Cocuzza, A. Selection of Aptamers That Bind to bFGF Tetrahedron Letters, (1989) 30:6287 6291). A 1M scale synthesis yielded 60 nmole of HPLC-purified single A control matrix comprised heparin-agarose 0.2 ml equili stranded randomized DNA. Each strand consisted of defined brated with selection buffer. Aptamer DNA (Round 1 DNA) primer 19-base and 20-base sequences at both the 5' and 3' was obtained by incubating a 0.2 mL support with 6.2 nmole ends of the strand, respectively, and a random region 60 of bFGF for one hour at room temperature. The resulting 10 bases in length in the center of the oligomer to generate a column was washed with 10x0.5 mL selection buffer. The pool of 99-mers with the following sequence (N=G.A.T or bFGF column was treated with 3x0.5 mL in selection buffer C): containing 2M NaCl. The second fraction of elution reflected the true eluted material. Aptamer DNA (Round 1 DNA) from bFGF peak was 15 brought to 0.1% SDS concentration and 20 mM ethylene diaminetetraacetic acid (EDTA), vortexed and extracted DNA 19 and 20-mers with the following sequences were with a mixture of 180 L phenol and 180 L chloroform. The used as primers for PCR amplification of oligonucleotide volume reduced to about 250 L. The resulting material was sequences recovered from selection columns. The 5' primer 20 sequence was 5' TGG-CAG-ATG-ACC-CGA-ATT-C (19 diluted with 0.5 mL and chromatographed on a NAP5 mer) 3' and the 3' primer sequence was 5' biotin-0- column in TE buffer. A 1 mL volume sample was collected AACATTTGAGCACTGGATCC (20 mer) 3". The biotin and the volume was reduced by repeated n-butanol extrac residue was linked to the 5' end of the 3' primer using tion to a final volume of 0.3 mL. Glycogen (20g) was added commercially available biotin phosphoramidite (New and the solution adjusted with 0.25M NaCl and precipitated 25 England Nuclear. Cat. No. NEF-707). The biotin phosphora with 0.9 mL absolute ethanol at -70 C. midite is incorporated into the strand during solid phase Approximately 2.59 pmole of template eluted and 25% DNA synthesis using standard synthesis conditions. was used in the PCR. B. Isolation of Factor X Aptamers Using Factor X Immo Two PCR amplification was conducted similar to bilized on a Lectin Column Example 5C with 501 template 97-mer, 201 10x-buffer (100 30 A pool of aptamer DNA 99 bases in length was synthe sized as described in Example 22-A, and then PCR mM Tris Cl, 500 mM KCl, 1.5 mM MgCl), 8 L dNTP amplified to construct the initial pool of about 10" different (25x), and 1 Leach of -'P dATP, dCTP and dGTP (80 Ci molecules. An aliquot of the enzymatically synthesized total), 3 L Taq I Polymerase (15 units), 3 L 5'-primer (1 DNA was further amplified in the presence of -P-dNTPs to nmole), 2.4 L 3'-primer (1 nmole). generate labeled aptamer to permit quantitation from column A control was also performed without template aptamer. 35 fractions. PCR was conducted for 18 rounds, concluding with a final A Factor X column was prepared by washing 1 mL (58 10 minute extension at 72 C. The resulting material was nmole) agarose-bound concanavalin A ("Con-A”) (Vector transferred to a 1.5 mL Eppendorf tube and extracted with Laboratories, cat, no. AL-1003) with 20 mM Tris-acetate 800 L of n-butanol. The resulting 100 L sample was applied buffer (pH 7.4) containing 1 mM MgCl2, 1 mM CaCl, 5 to a NICKTM column equilibrated in 100 mM Tris/100 mM mM KCl and 140 mM. NaCl (the "selection buffer") (4 NaCl. The second 400 L fraction was collected. Blank washes with 10 mL each). 1 mL of settled support was then retained <1.5% of the template and was not further utilized. incubated overnight at 4 C. in 10 mL selection buffer The resulting material was also applied to 400 Lavidin containing 368 ng (6.24 nmole) Factor X (Haematologic agarose column in the same 100 mM Tris/100 mM. NaCl Technologies Inc, Cat No. HCXA-0060). After shaking buffer. The column was washed three times with 1.5 mL of 45 overnight at 4 C. to permit Factor X binding to the Con-A high salt followed by 0.8 mLTE. The desired compound was beads, the mixture was briefly centrifuged and the superna removed and the column was treated with 0.8 mL of 0.15N tant removed. The beads were resuspended in fresh selection NaOH. The first 200 L fraction was discarded and the buffer and transferred to a column which was then washed remaining volume was collected, neutralized with glacial with selection buffer (5X1 mL). A column containing 1 mL acetic acid, and reduced by n-butanol extraction to 2001 50 of settled beads had a void volume of approximately 300 L. final volume. This material was chromatographed on A control Con-A column was prepared by adding 1 mL of 2-NICKTM columns equilibrated in 10 mM Tris; pH 7.4. The settled support to a column followed by 5 washes with 1 mL recovered material was heated at 95 C. for 5 minutes and of selection buffer per wash. slowly cooled to room temperature for 1 hr. to give 550 Prior to application of the aptamer DNA pool to Con-A pmole of the desired aptamer for bFGF. 55 columns, the DNA was heated in selection buffer at 95 C. for 3 minutes and then cooled to room temperature for 10 Example 22 minutes. The pool, consisting of 100 pmole DNA in 0.5 mL selection buffer, was then prerun on the control Con-A Selection of Aptamers that Bind to Factor X column at room temperature to remove species that bound to An improved selection process for obtaining aptamers the control support. Three additional 0.5 mL aliquots of that bind to Factor X was used to obtain specific binding selection buffer were added and column fractions 2, 3 and 4 species. Base sequences from the random region of indi (0.5 mL each) were pooled and then reapplied to the column vidual DNA clones is shown in FIG. 6. The protocol used is twice. The DNA in 1.5 mL selection buffer was then similar to that described in Example 5 but differs by utilizing recovered. Approximately 12% of total input cpm were DNA molecules with a 60 base random region instead of a 65 retained on the column, 30 base random region. Details of the selection are as The recovered DNA was then applied to a Con-A-Factor follows. X column as a 0.5 mL aliquot followed by a 1.0 mL aliquot. 5,756,291 123 124 Flow-through was retained and reapplied to the column 5.5 L of glacial acetic acid. The neutralized fractions were twice. DNA added to the column on the final application was reduced to 200 Lby speed vacuum or butanol extraction and left on the column for 1 hour at room temperature. The the nucleic acids were exchange over a NICKTM column. A column was then eluted with 0.5 mL aliquots of selection 5 L sample was removed for quantitation and analytical buffer. 0.5 mL fractions were collected and radioactivity was PAGE. The resulting amplified Round 1 Pool was applied to determined in each fraction. Radioactivity in eluted fractions a new Con-A-Factor X column as in Example 22-B to obtain 7 through 12 were low and relatively constant. After recov Round 2 aptamers. ery of fraction 12, the column was washed with 0.5 mL D. Characterization of Round 1 Through Round 7 Factor X aliquots of 0.1M -methyl-mannoside (Sigma Cat. no. Aptamers Obtained from Selection on Lectin Columns M-6882) in selection buffer to elute the bound Factor X Seven rounds of Factor Xaptamer selection and ampli along with Factor X-bound aptamers. Fractions 13, 14 and fication were carried out using Con-A-Factor X columns as 15 showed a significant peak of Factor X protein level, as in Examples 22-B and 22-C. As shown in Table 13, the determined spectrophotometrically by Bradford protein -methyl-mannoside eluate in fractions 14, 15 and 16 con stain (BioRad, Cat No. 500-0006). 0.090% of the input DNA tained a maximum of about 18% of input DNA at selection eluted in these two fractions. 15 round 7 using the described conditions. Aptamer DNA (Round 1 DNA) was recovered from the Factor X by phenol extraction (2x0.5 mL). The aqueous TABLE 13 phase volume was reduced to about 250 L by n-butanol % DNA eluted by % DNA bound to extraction. Aptamer DNA was precipitated on dry ice using -methyl-mannoside control support 3 volumes of ethanol and 20 g of glycogen as a carrier. The 09 12% DNA was pelleted, washed once in 70% ethanol and then 2 134 6% died, 3 513 9.5% C. Amplification of Factor X Selected Aptamers 4. 1.3 6.2% Round 1 DNA from Example 22-B was resuspended in 5 3.7% 2.3% 100 L of HO and amplified by PCR. A 200 L PCR reaction 25 6 849, 3.2% consisted of the following: 100 L template 99-mer DNA 7 17.9% 2.7% (approximately 0.05 pmoles); 20 L 10X buffer (100 mM *0.1M -methyl-mannoside in selection buffer was added beginning at fraction Tris-Cl (pH 8.3), 500 mM KCl 20 mM MgCl); 32 L 13 in each elution, and fractions 14 and 15 and 16 were retained and the DNA amplified. Due to slow leeching of Factor X from the column, DNA bound dNTP's (5 mM conc total, 1.25 mM each dATP, dCTP, to Factor X could also be seen in earlier fractions in rounds 6 and 7. DGTP, and dTTP); 20 L primer 1 (biotinylated 20-mer, 30 50M); 20 L primer 2 (19-mer, 50M); 6 L -'P-dNTP's After amplification, approximately 5 picomoles of radio (approximately 60 Ci); and 4 L Taq IPolymerase (20 units). labeled round 7 aptamer DNA was analyzed for specificity The reaction was covered with 2 drops NUJOL mineral oil. in a filter binding assay. In this assay, nitrocellulose filters (1 A control reaction was also performed without template cm diameter) pre-washed and also presoaked in selection aptamer. 35 buffer overnight at 4C. was used in 100L of selection buffer Initial denaturation was at 94 C. for 3 minutes, but (containing 30 nM to 3 M Factor X) was incubated at room subsequent denaturation after each elongation reaction temperature for 10 minutes with: (1) 96-mer (IIa clone 29); lasted 1 minute. Primer annealing occurred at 56 C. for 1 (2) unselected DNA; (3) Round 7 aptamer DNA. The minute, and elongation of primed DNA strands using the Taq mixtures were applied to the filters by vacuum. The filters polymerase ran at 72 C. for 2 minutes, with 5-second were then washed with 1.0 mL of selection buffer at 37 and extensions added at each additional cycle. The final elon radioactivity was counted to determine the amount of DNA gation reaction to completely fill in all strands ran for 10 that was retained as a Factor X complex. The results are minutes at 72 C. and the reaction was then held at 4 C. shown in FIG. 4. 24 rounds of Taq polymerase elongation were carried out Unselected DNA did not show significant binding to the in order to amplify the selected aptamer DNA. After the 45 Factor X while selected aptamer DNA bound to Factor X. reactions were completed, the aqueous layer was retrieved Binding results show specific Factor X binding. Factor X and any residual NUJOL oil was removed by n butanol inhibition was determined by conventional commercial extraction, reducing the volume to 100 L. A sample may be assay using Russell Viper Venom (Sigma) with the results removed from each of the aptamer and control reaction for set forth in FIG. 5 for the 99-mer pool of Example 22. quantitation and analytical PAGE. The amplified aptamer 50 The Viper Venom assay was carried out in a 250 L well pool (100 L) was fractionated over a Nick column (G-50 of a microtiter plate. Factor X, (2 pM final concentration) 47 Sephadex, equilibrated with 3 mLTE buffer (10 mM Tris ng of protein is incubated with or without DNA (21.25 uM) HCl (pH 7.6), 0.1 mM EDTA)) to remove unincorporated in 40 L of Selection Buffer at 37 for 2-3 min. 10 L of NTPS. primers, and salt. 400 L of TE buffer was then added RVV/CaCl (0.066 mg/mL in 0.133M CaCl) is added and to the column and the DNA pool was eluted from the column 55 incubated at 37 for 60 sec. 50L of 0.5 mM Spectrozyme FXa with an additional 400 L using TE buffer. (Asample may be No. 222 (American Diagnostica) in 45 nM Tris. Cl pH 8.3 removed from the eluent for quantitation and analytical and allowed to react for 120 sec at 37. The reaction was PAGE.) The eluent (400 L) was loaded on an avidin agarose stopped with 10 L of 20% acetic acid. column (Vector Laboratories, Cat. No. A-2010) (500 L Tissue factor has minimal procoagulant activity prior to settled support, washed with 3x1 mLTris/NaCl buffer (0.1M replipidation. The apoprotein was relipidated by incubation Tris, 0.1M NaCl, pH 7.5)). Approximately 90% of the at 37 C. for 30 min in a relipidation mixture containing 800 loaded radioactivity remained on the column. The column L of TBSA (50 mM tris HCl, pH 7.5, 100 mM. NaCl, 0.1% was washed with Tris/NaCl buffer (4x400 L) and then the BSA) and 50L of a 5 mg/ml solution of phosphatidylcholine nonbiotinylated strand was eluted with 0.15N NaOH (4x300 in 0.25% deoxycholic acid and 25 L of 100 mM CdCl2. The L fractions). More than 45% of the radioactivity on the 65 relipidated TF was then incubated for 5 minutes at 37 C. in column eluted in the last three fractions. These fractions 50 L of TBSA containing Factor Xincubated with DNA and (900 L) were combined and neutralized with approximately Factor VII and 100 L of 25 mM CaCl. Fifty microliters of 5,756.291 125 126 the FXa substrate was then added and incubated for an these aptamers and the use of the aptamers in the detection additional 10 min at 37 C. The reaction was stopped by the and isolation of such substances. Although preferred addition of 50% acetic acid and absorbance at 405 nm was embodiments of the subject invention have been described then detected. in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the Example 23 appended claims. Selection of Aptamers that bind to ICAM-1 TABLE 1. Five peptides (between 17 and 19 residues) encompassing the active domains of ICAM-1 were selected and obtained 10 A. Extracellular Proteins: from commercial sources. Each peptide was 80% pure. lipoprotein lipase Peptide sequences used were: lecithinlist-cholesterol acyl transferase apolipoprotein A-1 apolipoprotein II (25)YDQPKLLGIETPLPKKEL (43) apolipoprotein IV (35) TPLPKKELLLPGNNRKVYE (53) 15 apolipoprotein B-48 (44) LPGNNRKVYELSNVQEDSQ (62) apolipoprotein B-100 (66) YSNCPDGQSTAKTFLTVY (83) apolipoprotein CI (162)YELDLRPQGLELFENTS (178) apolipoprotein CII apolipoprotein CITI Numbers in parentheses indicate the amino acid number apolipoprotein D from the amino terminus of ICAM-1. apolipoprotein E Each peptide was dissolved in sterile water as a concen insulin trated solution. Each peptide at 10M was incubated with a insulin-like growth factors I and II angiotensin I DNA pool consisting of 60 bases of random DNA flanked by angiotensin II PCR primer sequences in 50 L selection buffer. The DNA renin was radiolabeled with 'P to permit localization of the DNA 25 angiotensin converting enzyme on gels described below. DNA that specifically interacted atrial matriuretic peptide immunoglobulin IgA constant region with the peptides was separated from the free DNA by innunoglobulin IgG constant region electrophoresis in a low salt (50 mM HEPES, pH 7.5, 1 mM immunoglobulin IgE constant region MgCl2, 2 mM KCl), 4 or 6% polyacrylamide gel or a 2.5% immunoglobulin IgM constant region agarose gel (Seakem, ME). DNA molecules that migrated immunoglobulin light chain kappa immunoglobulin light chain lambda more slowly, i.e. gel shifted, than the free pool DNA were immunoglobulin IgGFc portion isolated from the gel and subjected to 20 rounds of PCR immunoglobulin IgM Fo portion amplification. DNA was recovered from acrylamide gels by immunoglobulin IgE Fc portion soaking gel regions containing the desired DNA overnight at amyloid protein room temperature in 15 mL of 0.3M NaOAc followed by 35 beta-amyloid protein substance P ethanol precipitation. DNA was recovered from agarose gels leu-enkephalin using Mermaid TM according to manufacturer's instructions met-enkephalin (Bio 101). Approximately 1% of the molecules migrated somatostatin more slowly than the bulk pool. After four rounds of interleukin-1 interleukin-2 selection, a DNA species was selected for that migrated interleukin-3 more slowly than the starting pool even in the absence of interleukin-4 peptide (presumably due to binding to the gel matrix) along interleukin-5 interleukin-6 with DNA species that were selected due to specific ICAM interleukin-7 peptide binding. A negative selection process was therefore interleukin-8 introduced to remove these DNAs. Round 4 DNA without 45 interleukin-9 added peptide was fractionated as before and the fast migrat interleukin-10 interleukin-11 ing DNA was isolated and reintroduced into the peptide interleukin-12 selection process. The negative selection efficiently removed interleukin-13 the presumptive gel matrix binding species. colony stimulating factor-macrophage SO colony stimulating factor-granulocyte Example 24 colony stimulating factor-macrophagelgranulocyte erythropoietin myelin basic protein Selection of Aptamers that bind to VCAM-1 and E carcinoembryonic antigen Selectin collagen type I collagen type I Aptamers that bind specifically to VCAM-1 and 55 collagen type III E-Selectin are obtained and characterized using methods collagen type IV essentially as described above for thrombin, Factor X or collagen type W ICAM-1. VCAM-1 or E-Selectin protein or active domain vitronectin fibronectin peptide is used with lectin columns or in gel shift selections fibrinogen to obtain specifically binding aptamers. The selection pro albumin cess is conducted using a combination of lectin columns aminopeptidase with gel shift selections or by using either method alone. amylase avidin Thus, methods for obtaining aptamers that specifically B-cell growth factor bind serum proteins such as thrombin and Factor X. growth Bence-Jones protein factors such as FGF, adhesion molecules such as ICAM-1, 65 prothrombin (Factor II) eicosanoids, kinins such as bradykinin, and cell surface thrombin (Factor IIa) ligands are described, as well as the therapeutic utility of 5,756.291 127 128

TABLE 1-continued TABLE 1-continued tissue factor (Factor II) fibrinopeptide B proaccelerin (Factor W). filaggrin accelerin (Factor Wa) follicle-stimulating hormone proconvertin (Factor VII) follicle-stimulating hormone releasing hormone antihemophiliac factor (Factor VIII) gastrin releasing peptide Christmas factor (Factor DX) growth hormone Stuart factor (Factor X) glucagon plasma thromboplastin antecedent (Factor X) leutinizing hormone Hageman factor (Factor XET) 10 leutinizing hormone releasing hormone Fibrin-stabilizing factor (Factor XIII) human menopausal gonadotropin prekallikrein prolactin high molecular weight kininogen chorionic gonadotropin bradykinin growth hormone releasing hormone kinins henosiderin calcitonin 15 placental lactogen carboxypeptidase A inhibia carboxypeptidase B kalikrein carboxypeptidase C acid keratin catalase vimentin ceruloplasmin desmin cholinesterase 2O glial fibrillary acidic protein chymotrypsin leukokinin lipase leupeptin amylase luciferase collagenase melanin complement protein Cliq melanotropin complement protein Cir2 melato in complement protein Cls2 25 melanotropin release inhibiting homone complement protein C2 nerve growth factor complement protein C2a oxytocin complement protein C2b vasopressin complement protein C3 convertase neurophysin complement protein C3 neurotensin complement protein C3b 30 beta-endorphin complement protein C4 adrenorphin complement protein C4a dynorphin complement protein C4b alpha neoendorphin complement protein. C5a phospholipase A2 complement protein C5 papain complement protein C5b 35 plasmin complement protein C5 convertase acidic alphal glycoprotein complement protein C6 alpha 1 lipoprotein complement protein C7 alpha trypsin inhibitor complement protein C8 beta 1 lipoprotein complement protein C9 hemopexin complement protein lytic complex 40 alpha 1 antitypsin adrenocorticotropic hormone transferrin corticotropin releasing hormone plasminogen pepsin A platelet derived growth factor (alpha and beta) pepsin B acidic fibroblast growth factor pepsin C basic fibroblast growth factor trypsin somatotropin release inhibiting hormone elastase somatotropin releasing hormone enterokinase superoxide dismutase leucine aminopeptidase thymosin aminotripeptidase thyrotropin dipeptidase thyrotropin releasing hormone prolidase alpha fetoprotein prolinase 50 tumor necrosis factor-alpha alpha-glucosidase tumor necrosis factor-beta SS vasoactive intestinal opeptide maltase von Wilebrand factor beta-galactosidase tissue plasminogen activator oligo glucosidase gondatropin releasing hormone lipase 55 parathyroid hormone phospholipase A and B antithrombin I cholesterol esterase protein C monoacylglycerol lipase protein S carboxylic acid esterase activated protein C alkaline phosphatase interferon alpha elastin 60 interferon beta myelin protein Al interferon ganna proenkephalin ferrit enteropeptidase haptoglobin L-asparaginase HDL E-asparaginase LDL epidermal growth factor WLDL fibrin 65 TGF (alpha and beta) fibrinopeptide A Steel Factor (stem cell growth factor)

5,756.291 13 132

TABLE 1-continued TABLE 1-continued IL-4 receptor hemagglutinin (HA) Parathyroid hormone receptor neuraminidase (NA) GnRH receptor nucleoprotein (NP) CSF-M receptor M1 and M2 proteins CSF-GM receptor NS1 and NS2 proteins CSF-G receptor Hepatitis B Erythropoietin receptor Complement receptor Envelope (surface antigenP proteins (including pre-S1, Cb receptor O pre-S2 and S) EGF receptor Nucleocapsid (core) proteins Follicle stimulating hormone receptor P-gene product Follicle stimulating hormone releasing hormone receptor X-gene product Growth hormone receptor Cytomegalovirus Glucagon receptor Leutinizing hormone receptor 15 Immediate early (alpha) gene products (including IE1 and Leutinizing hormone releasing hormone receptor EE2) Growth hormone releasing hormone receptor Early (beta) gene products (including DNA poll plaC, DBP52 Nerve growth factor receptor EDBP 140) Melanotropin release inhibiting hormone receptor Late (gamma) structural gene products Platelet derived growth factor receptor (alpha and beta) Herpes Simplex Virus Fibroblast growth factor receptor (i and 2) Somatotropin release inhibiting hormone receptor Somatotropin releasing hormone receptor ribonucleotide reductase Thyrotropin receptor virus-encoded envelope glycoproteins Thyrotropin releasing hormone receptor Epstein-Barr Virus Tumor necrosis factor alpha receptor Tumor necrosis factor beta receptor immediate early gene products (including ZLF1 protein and Complement C3a receptor 25 RLF1 protein) Complement C5a receptor early gene products (including SMLF1, MRF1, ALF2, HRF1, Complement C3b receptor ribonucleotide reductase, thymidine kinase XLFD Complement CR2 receptor virus-encoded glycoproteins Complement CR3 receptor lipopolysaccharides (from gram negative or grain positive CSF-1 receptor bacteria) GMCSF receptor 30 SLF receptor botulinum toxin flg oncogene protein diphtheria toxin c-ros oncogene protein cholera toxin erb-B2 oncogene protein endotoxin trk-B oncogene protein D. Intracellular Targets (proteins/lipids/Enzymes trk oncogene protein 35 c-fems oncogene protein Lipids c-kit oncogene protein erb-B oncogene protein fatty acids HER-2/neu oncogene protein glycerides kit oncogene protein glycerylethers C. Wirus and Bacterial Targets phospholipids sphingolipids HIV-1/HTW-2 steroids fat soluble vitamins reverse transcriptase (including RNAse H) glycolipid protease phospholipids lecithins gag proteins (including pl.p24, pl5) 45 phosphatidic acids (cephalins) tat protein sphingomyelin rev protein plasmalogens nef protein phosphatidylinositol wif protein phosphatidylcholine vpr protein phosphatidyl serine vpu protein 50 phosphatidyl inositol envelope proteins (including gp120, gp41) diphosphatidylglycerol HTLV-II oleic palmitic gag proteins (including gp24, gp19, gp15) protease stearic acids pol (including reverse transcriptase and RNAse H) linoleic acid envelope genes (including gp46 and gp41) 55 acyl-coenzyme A tax phosphoglyceride e phosphitidate Human papillomaviruses E7 protein retinoic acid retinoids E6 protein lipoprotein A E6* protein proteolipid E4 protein sphingolipids E1 proteins sphingosine El-E4 proteins ceramides E2 proteins cerebrosides capsid proteins (L1 and L2) gangliosides influenza A and B sphingomyelins 65 terpenes polymerase proteins (including PA, PBI, and PB2) sesquiterpenes 5,756,291 133 134

TABLE 1-continued TABLE 1-continued diterpenes 1-acylglycerol-3-phosphate acyltransferase triterpenes 3-b-hydroxy-steroid dehydrogenase(EC5.3.3.1) tetraterpenes 5 3-hydroxybutyrate dehydrogenase steroids 3-ketothiolase cholesterol 5'-nucleotidase cholesterol esters 8-oxoguanosine deglycosylase cholic acid 11b-hydroxylase (EC 1.14.15:4) phosphatidylcholine 18-hydroxylase estrogen 10 21-steroid hydroxylase(EC 1119910) testosterole 2,3-oxidosqualenelanosterol cyclase androgens 24.28-sterol reductase 2-keto-3-deoxyoctanoate a-actin Intracellular proteins a-Imanosidase a-melogenin p53 15 a-tubulin pRB retinoblastoma gene product) acetolactate synthase methemoglobin acetylcholinesterase hemoglobin A acetylglucosaminyl transferase hemoglobin Al acetyl spermine deactylase hemoglobin A2 acetyl transacylase hemoglobin Barcelona 20 acetyl-CoA carboxylase hemoglobin Barts acetyl-CoA malate citrate synthase hemoglobin Beth Isreal acid phosphatase hemoglobin Bunbury acid protease hemoglobin Cochin-Port Royal aconitase hemoglobin Cowtown actin hemoglobin Cranston adenosine dearninase hemoglobin Creteil 25 adenosylhomocysteine hemoglobin D adenosylmethionine decarboxylase hemoglobin D-Los Angeles adenylate cydase hemoglobin D-Punjab adenylate deaminase hemoglobin F hemoglobin Gower adenylsuccinate hemoglobin Hammersmith 30 adenylsuccinate synthase hemoglobin Hiroshima alanine aminotransferase hemoglobin Indianapolis alcohol dehydrogenase hemoglobin Kansas aldolase hemoglobin Kariya adose reducase hemoglobin Kempsey alkaline phosphatase hemoglobin Kenya 35 amidophosphodbosylanine transferase hemoglobin Lepore AMP phosphodiesterase hemoglobin M amyloid by A4 protein hemoglobin M Hyde Park amyloid precursor protein hemoglobin M Iwate ankarin hemoglobin M. Saskatoon arginase hemoglobin Nancy 40 argininosuccinate lyase hemoglobin Philly argininosuccinate synthetase hemoglobin Quong Sze aTOnatase hemoglobin Ranier aryl sulfatase hemoglobin Raleigh aspartate aminotransferase hemoglobin S aspartate transcarbamoylase hemoglobin Sealy ATP diphosphohydrolase hemoglobin Seattle 45 ATPase hemoglobin St. Louis b-actin hemoglobin St. Mande b-glucuronidase hemoglobin Titusville b-glycerophosphatase hemoglobin Torino b-ketoacyl-ACP reductase hemoglobin Wayne b-ketoacyl-ACP sythetase hemoglobin York 50 b-spectrin hemoglobin Zurich b-tropomyosin src oncogene protein b-tubulin abl oncogene protein C5a inactivation factor met oncogene protein calcitoin Ha-ras oncogene protein calmodulin Ki-ras oncogene protein 55 calpain. I N-ras oncogene protein calreticulin fps oncogene protein carbamoyl-phosphate synthetase mos oncogene protein carbonic anhydrase raf oncogene protein casein kinase 1 pim oncogene protein casein kinase 2 crk oncogene protein 60 catalase dbloncogene protein catechol methyltransferase rel oncogene protein cathepsin yes oncogene protein cathepsin B and L fgroncogene protein cdc. 2 p.34 L-myc oncogene protein cdc. 10 int-1 oncogene protein cdc. 13 p60 ets oncogene protein 65 cdc. 25 p8O bcl-2 oncogene protein chaparonin 5,756.291 135 136

TABLE 1-continued TABLE 1-continued cholesterol esterase mixed function oxygenase cholesterol monooxygenase myloperoxidase citrate synthetase myofilament clathrin myristoyltransferase collagenase N-acetylglucuronidase connective tissue activating peptide NaKATPase core protein NAD-dependent sterol 4-carboxylase cortisol dehydrogenase NADase cyclin A and B 10 NADPH-dependent 3-oxosteroid reductase cyclophilin nexin cytidine deaminase nucleolar protein B23 cytidylate deaminase nucleoside diphosphate kinase cytochrome C peroxidase ornithine aminotransferase cytochrome P450 ornithine carbamoyltransferase cytosine methyl transferase 15 ornithine decarboxylase defensin orotate decarboxylase diacylglycerol acyltransferase orotate phosphoribosyl transferase dihydrofolate reductase peptidyl prolyl dihydrouraci dehydrogenase peptidylamidoglycolate lyase dihydroorotatase phenylalanine hydroxylase dihydroorotate dehydrogenase phosphatidate phosphatase dioxygenase 20 phosphoenolpyruvate carboxykinase dopamine monooxygenase dynenin phosphoglucokinase elastase phosphoglucomutase elastin elongation factor Tu phosphoglyceromutase endo-rhamosidase 25 phospholipase A2 enolase phospholipase C enoyl-ACP hydratase phospholipase CGl enoyl-ACP reductase phospholipase D fatty acid synthetase phospholipase S ferritin phosphoribomutase ferrodoxin phosphoribosylphosphate transferase fructose bisphosphate aldolase plasminogen activator inhibitor funarase platelet factor 4 GABA aminotransferase porin galactosidase pRb rentinablastoma gene product gelatinase p idi gelsolin 35 prostaglandin synthase glucophosphate isomerase C glucosylceramide galactosyl transferase purine nucleoside phosphorylase glutaminase pyruvate dehydrogenase glutamine phosphoribosylpyrophosphate aminotransferase glycerol phosphate acyl transferase ribonucleotide reductase glycerol phosphate dehydrogenase ribosephosphate pyrophosphate kinase glycinamide ribonucleotide transfomylase ricin tropoelastin GTP binding protein heavy meromyosin spectrin spermine synthase histaminase squalene epoxidase histidine decarboxylase squalene monooxygenase HSP 27 45 sterol methyltansferase suc 1 p13 hydroxy acyl CoA dehydrogenase succinyl CoA synthetase hydroxy steriod dehydrogenase superoxide dismutase hydroxy-methylglutaryl CoA cleavage enzyme tartrate dehydrogenase hydroxy-methylglutaryl CoA reductase thioesterase hydroxy-methylglutaryl CoA sythetase 50 thioredoxin hypoxanthine-guanine phosphoribosyl transferas thrombospondin EMP dehydrogenase thromboxane A2 synthetase indole lyase thymidylate synthetase inositol phosphate phosphatase transacylase isocitate lyase triosephosphate dehydrogenase kinin generating enzyme 55 triosephosphate isomerase lactate dehydrogenase tRNA synthetase lactoferrin tropomyosin lamini tryptophan synthase leukocyte elastase lipocortin tubulin lipoxygenase long chain fatty acid CoA ligase ubioquinone reductase lysozyme uridine monophosphate kinase Inajor basic protein urokinase type plasminogen activator malate dehydrogenase vitamin Kreductase malate synthase wee -1 gene product malonyl transacylase xanthine dehydrogenase mannosidase 65 xanthine oxidase methionine adenosyltransferase xylosyl transferase 5,756.291 137 138

TABLE 1-continued TABLE 1-continued E. Small Molecules and Other Compounds corticosterone cortisol 2-phosphoglycerate 5 cortisone 3-hydroxy acyl-CoA coumarin 3-phospho-5-pyrophosphomevalonate creatine 3-phosphoglycerate creatinine 3-phosphohydroxypyruvate CTP 3-phosphoserine cyanocobalamin 5-alpha-dihydrotestosterone 10 cyclic AMP 5-phospho-beta-ribosylamine cyclic CMP 5-phosphoribosyl 1-pyrophosphate cyclic GMP 5-phospho-alpha-ribosyl-l-pyrophosphate cyclic TMP 5-phosphoribosyl-4-carboxamide-5-aminoimidazole cystathionine 6-benzylaminopurine cytnidine 17-hydroxyprogesterone 15 cytochrome acetominophen D- Se aceyt-coenzyme A D-Fructose acetylcholine D-Galactosamine acetylsalicylic acid D-glucose adenine D-Glucuronic acid adenosine 2O dADP ADP dAMP aflatoxin B dATP aflatoxin G1 dCDP aflatoxin Ml dCMP aldosterone dCTP aliantoin delta-4-androstenedione allodeoxycholic acid 25 deoxyadenosylcobalamin allopurinol deoxycholic acid alpha ketoglutarate diGDP alphabeta-dihydroxy-beta-methylvalerate dGMP alpha-aceto-alpha-hydroxybutyrate dGTP alpha-amino-beta-ketoadipate dihydroorotate alpha-bungarotoxin 30 dihydroxyphenylalanine alpha-carotine diphosphoglycerate alpha-keto-beta-methylvalerate dopanane alpha-ketoglutarate dTDP alpha-ketobutyrate dTMP alpha-ketoglutarate dTTP amiloride 35 dUDP aminopterin dUMP AMP dUTP amylopectin eosinophil chemotactic factor of anaaphyaxis-A amylose epinephrine anti-diuretic hormone estriol antipyrine 40 esdone arachidic acid ethynylestridiol arachidonic acid FAD arecoline farnesyl pyrophosphate arginine fatty Acyl-s-CoA argininosuccinate ferrodoxin ascorbic acid FMN aspartate semialdehyde 4.5 FMNH2 aspartyl phosphate folic acid ATP fructose 2,6-diphosphate atropine fructose bacitracine fructose 1,6-diphosphate benztropine fructose 6-phosphate beta-caratine 50 Fructosel,6-diphosphate betamethazone fumarate bilirubin galactose billiverdin galactose biotin GalNAC carbachol gama-aminolevulinate carbamoyl phosphate 55 gamma-carotene carboline gastric inhibitory protein carinitine gaunidinoacetate CDP GDP cholesterol gentalnycin cholic acid glucosamine chorismic acid 6 ghucosamine 6-phosphate cis aconitate glucose citrate glucose 1,6-diphosphate citrulline glucose 1-phosphate CMP glucose 6-phosphate cocaine Glutamate codeine glutamate semialdehyde Coenzyme Q 65 glutaryl-CoA coenzyme A glutathione 5,756,291 139 140

TABLE 1-continued TABLE 1-continued glyceraldehyde 3-phosphate glycerol. 1-phosphate E.hosph G tini e glychocholate 5 p ysos gmine glycine pilocarpine glyoxylate piperidine GMP pirenzipine GIP plastoquinone guanine hemichohne O platelet-activating factor histamine porphobilinogen homogentisate pregnenolone homoserine progesterone

E.ydroxypro prolinanide indole 15 propionyl-CoA inosine prostaglandin D2 inositol protoporphyrin DX inositol phosphate pteridi intermediate molecular weight eosinophil chemotactic yridoxal factor of isocitrate pyr isopentenyl pyrophosphate 2O pyridoxal phosphate L-alanine pyridoxal phosphate E. pyridoxamine L-aspartic acid pyridoxamine phosphate L-aspartic acid pyrodoxine L-azoserine pyroglutamic acid L-cysteine 25 pyrophosphate -Fucose rrollidine L-glutamic acid py L-glutamine pyrroline-5-carboxylate L-histidine pyrrolizidine L-isoleucine inplizidine L-leucine 30 quip w L-lysine quinuclidinylbromide L-malate RGD peptide L-methionine riboflavin L-phenylalanine r L-proline L-serine 35 sadenosylhomocysteine L-threonine s-adenosylmethionine L-typtophane scopolamine L-tyrosine serotonin lanosterolL-valine slownreacting substance of anaphylaxis- leukotriene B4 40 squalene leukotriene C4 suberyldicholine leukotriene D4 succinate lipoicleukotrienes acid succinyl-CoA- luciferin taurocholate Negi:malonate testosteoletetrahydrofolic acid OC methotrexate thiamine methylenetetrahydrofolate thioredoxin mtethylmalonyl-CoA thromboxane A2 mevalonate thromboxane B2 mevalonate-5-phosphate 50 muscarin thymine N-Formylmethionine tropane NAD ubiquinol NADH ubiquinone NADP UDP NADPH 55 neostignine UDP-galactose nicotinanide UMP nicotine uracil nicotinic acid norepinephrine-- sea ornithine uric acid oxaloacetate 60 UTP oxotrernorine witamin A p-benzoquinone vitamin D pancuronium a pantotheric acid vitamin E para-aminobenzoic acid 65 vitamin K phosphenolpyruvate 5,756.291 141 142

SEQUENCE LISTING

( t ) GENERAL INFORMATION: ( i i i NUMBER OF SEQUENCES: 181

( 2) INFORMATION FOR SEQID NO:1: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID NO:1: Cy s Pro Arg Th r A s in Arg 5

( 2) INFORMATION FOR SEQID NO:2: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE:amino acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:2: A s p Pro A rig Le u I le A sp 1 5

( 2) INFORMATION FOR SEQID NO:3: ( i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 9amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQED NO:3: A rig Pro Pro G 1 y Ph e Sier Pro Phe Arg 1. 5

( 2) INFORMATION FOR SEQID No.4: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(20,") (D OTHER INFORMATION: note= "This is a 60 nucleotide stretch of random sequences." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:4: CGT ACG GT C G A CGCTAG CNC ACG T G GAG C T C G GATCC 3 7

( 2 INFORMATION FOR SEQID No.5: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 18 base pairs (B) TYPE: nucleic acid (CSTRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No.5:

C GT ACG GT C G A CGCTA G C 18 5,756.291 143 144 -continued

( 2) INFORMATION FOR SEQID NO;6: i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear i x FEATURE: (A) NAME/KEY: misc difference ( B ) LOCATION: replace(1,") ( D ) OTHER INFORMATION: inote= This position is biotin-O. G." ( x i SEQUENCE DESCRIPTION: SEQID NO-6:

GG AT C C : A G C T C CAC GT G 8

( 2) INFORMATION FOR SEQID NO:7: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQED NO:7: A CGC C G CG G T ACT AC GCNN NN 22

( 2 INFORMATION FOR SEQID NO:3: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH; 18 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE; (ANAME/KEY: Inisc difference ( B LOCATION: replace(1,") ( D ) OTHER INFORMATION: note= This position is biotin-T” ( xi ) SEQUENCE DESCRIPTION: SEQID NO:3: T C C C C C C C C A T GAAt GC G 8

(2) INFORMATION FOR SEQID No.9. ( i SEQUENCE CHARACTERISTICS: (A LENGTH: 22 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:9:

A G CG GCC GCT C T CTA GANN NN 22

( 2 INFORMATION FOR SEQID NO:10: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 18 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:to:

T C C C C GC GA GAA GAT C 3

( 2 INFORMATION FOR SEQID No:11: 5,756.291 145 146 -continued ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid C ) STRANDEDNESS: single ( D y TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:11:

GA GAA CNNNN

( 2) INFORMATION FOR SEQID NO:12: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference B ) LOCATION: replace(1,") ( D ) OTHER INFORMATION: note= "This position is biotin-T." ( i x FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(18.") (d) OTHER INFORMATION: fnote "This position is a thioate linkage."

( xi ) SEQUENCE DESCRIPTION: SEQID No:12:

T G C G G C G CCA GAA T G C G

( 2) INFORMATION FOR SEQID NO:13: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 37 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc-difference (B) LOCATION: replace(1,") ( D.) OTHER INFORMATION: note= "This position is biotin-T" ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(18, "y Id OTHER INFORMATION: note "This position is a thioate

( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(19,") (D) OTHER INFORMATION: note= This is a 60 nucleotide stretch of random sequences." ( x i ) SEQUENCE DESCRIPTION: SEQD NO:13: T G C G G C G CCA T GAA T G C GNT CTA GAA GA G C G G C C GCT

( 2) INFORMATION FOR SEQID NO:14: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(1," (D) OTHER INFORMATION: Enote= This position is PO3-A."

( i x ) FEATURE: (A) NAME/KEY: misc difference 5,756,291 147 148 -continued ( By LOCATION: replace(6,") D OTHER INFORMATION: note= This position is a 55 nucleotide stretch of randon sequences." x i SEQUENCE DESCRIPTION: SEQD NO:14:

NATCN

( 2) INFORMATION FOR SEQID NO:15: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 7 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single D TOPOLOGY: linear ( i x FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(1,") (D) OTHER INFORMATION: note- “This is a 17 nucleotide stretch of abasic residues."

( x i ) SEQUENCE DESCRIPTION: SEQID NO:15:

NCAA

2 INFORMATION FOR SEQID NO:16: ( i) SEQUENCE CHARACTERISTCs: (A) LENGTH: 2 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(1,") ( D.) OTHER NFORMATION: note= This is a biotin-17 nucleotide stretch of abasic residues." ( i ) SEQUENCE DESCRIPTION: SEQID NO:16:

NG

( 2 INFORMATION FOR SEQID NO:17: ( i) SEQUENCE CHARACTERISTICS: (A) LENGH: 39 base pairs (B) TYPE: nucleic acid (CSTRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEAURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(20,") ( D.) OTHER INFORMATION: Enote= This is a 30 nucleotide stretch of randora sequences." ( x i SEQUENCE DESCRIPTION: SEQID NO:17:

T C C C G A C CAA. G. CTTA N C GAA TT C C C GA GT CAGA 3 9

( 2) INFORMATION FOR SEQID No:18: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: stucleic acid ( CSTRANDEDNESS: single ( D TOPOLOGY: linear ( i SEQUENCE DESCRIPTION: SEQId No:18:

T C T C C G A C CAA TTA. 9

2 INFORMATION FOR SEQID NO:19: 5,756,291 149 150 -continued

( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 19 base pairs (B) TYPE: mucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(1,") { D ) OTHER INFORMATION: note= This position is biotin-O-T." ( x i ) SEQUENCE DESCRIPTION: SEQED No:19: TCT AGA C T C G A G GAATCG

( 2) INFORMATION FOR SEQID NO:20: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 37 base pairs { B ) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i x FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(19,") (d) OTHER INFORMATION: note= This is a 60 nucleotide stretch of randon sequences." ( x i y SEQUENCE DESCRIPTION: SEQID NO:20: CGT ACG GT C G A C C CTA CNC A CG G GAG C T C G GA C C

( 2) INFORMATION FOR SEQID NO:21: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:21;

CGTA CG G C G A C GCTA GC

( 2) INFORMATION FOR SEQID No:22: ( i SEQUENCE CHARACTERISTICs: ( A LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID No:22:

G GAT C C GA G C T C CAC GT G.

( 2) INFORMATION FOR SEQ ID No:23: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE; (A) NAMEKEY: Inisc difference (B) LOCATION: replace(20,") (D) OTHER INFORMATION:/note= "This is a 60 nucleotide stretch of random sequences." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:23:

A GAA. A CT CA A G CTT GCC GN A C C T GAA TT C GCC CTATAG 5,756.291 151 152 -continued

2 ) INFORMATION FOR SEQID NO:24: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 19 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(...") ( D ) OTHER INFORMATION: note "This position is biotit-O- C.”

( xi ) SEQUENCE DESCRIPTION: SEQID NO:24:

CTA AGGG C G AATTCACGT 9

( 2) INFORMATION FOR SEQID NO:25: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 19 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQD NO:25:

A GAA. TACT CA A G C T G C CG 9

( 2 INFORMATION FOR SEQID NO:26: ( . ). SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 36 base pairs ( B, ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID NO:26:

TAATA CGA C CAC TATA GGG AT C C G A G C C CA C C G 3 6

( 2 INFORMATION FOR SEQID NO:27: { i y SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQED NO:27:

CT CAC GT cis ACG A. GC

( 2 INFORMATION FOR SEQID No:28: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single { D TOPOLOGY: linear ( i x ) FEATURE: ( A NAMEKEY: misc difference B LOCATION: replace(3," D OTHER INFORMATION: Enote= This position is usually T and never C.

i x ) FEATURE: ( A NAME/KEY: nic difference ( B . LOCATION: replace07, "). ( D OTHER INFORMATION: inote= This position is Nz where N is usually T and never C, and z is an integer from 2 to 5. 5,756,291 153 154 -continued

( i x ) FEATURE: (A) NAME/KEY: misc difference ( B LOCATION: replace(10,") ( D.) OTHER INFORMATION:/note= This position is usually T and never C.' ( x i ) SEQUENCE DESCRIPTION: SEQID NO:28:

GGNT GGN G GN T G G

( 2) INFORMATION FOR SEQID NO:29: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs B TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:29:

GGTT G G T G T G GT T G G

( 2 INFORMATION FOR SEQID NO:30: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(15.17, ") { D ). OTHER INFORMATION:/note "These positions are linked by MEA." ( x i SEQUENCE DESCRIPTION: SEQID NO:30:

GGTT G G G T G GT TGG GT

( 2) INFORMATION FOR SEQID NO:31: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS; single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(13.15. ") (d) OTHER INFORMATION: inote= "these positions are thioate ( i. e. . , P (O)S) linked." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:31:

G G T G G T G T G GTT G. G.

( 2) INFORMATION FOR SEQID NO:32: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: mucleic acid ( C ), STRANDEDNESS: single (d) TOPOLOGY: linear ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(1.15, ") ( D ) OTHER INFORMATION:/note= "These positions are thioate ( i. e. P (OS) linked” ( xi ) SEQUENCE DSCRIPTION: SEQ d no:32: 5,756.291 155 156 -continued

G G T G G T G T G G T G G 5

( 2) INFORMATION FOR SEQID NO:33: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear { i t ) FEATURE: ( A ). NAME/KEY: Inisc difference (B) LOCATION: replace0?,") ( D.) OTHER INFORMATION: inote "This position is deoxyuridine." ( i , ) FEATURE: ( A NAMEKEY: misc difference ( B LOCATION: replace09, "). ( D.) OTHER INFORMATION: note= This position is deoxyuridine." ( xi ) SEQUENCE DESCRIPTION: SEQED NO:33:

GTT GNN G T G G 1 5

( 2) INFORMATION FOR SEQID NO:34: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 5 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3," ( D.) OTHER INFORMATION: Enote= This position is deoxyuridine." { i : ) FEATURE; ( A NAME/KEY: Inisc difference (B) LOCATION: replace(12," (D) OTHER INFORMATION: note= “his position is deoxyuridine." ( x i SEQUENCE DESCRIPTION: SEQID NO:34:

GGNT G T G T G GNT G G 5

( 2) INFORMATION FOR SEQID NO:35: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE; (ANAMEKEY: misc difference ( B LOCATION: replace(13, ") ( D. OTHER INFORMATION: inote= This position is 5-(1-pentynyl)tracil" ( x i SEQUENCE DESCRIPTION: SEQID NO:35:

G G T G G T G T G GTNG G 5

( 2) INFORMATION FOR SEQED NO:36: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid (c) STRANDEDNESS: single (D) TOPOLOGY: linear 5,756,291 157 158 -continued

( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3.4. ") ( D ) OTHER INFORMATION: Anote= "This is a formacetal linkage." ( x i SEQUENCE DESCRIPTION: SEQID NO:36:

GGTT G G T G T G GT T G G 5

( 2) INFORMATION FOR SEQID No:37: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(12.13, ") (d) OTHER INFORMATION: inote= "This is a formacetal linkage." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:37:

G GT T G G T G T G GT T G G 15

( 2) INFORMATION FOR SEQID No.38: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear { i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3.4, "). { D ) OTHER INFORMATION: inote= "his is a formacetal linkage." ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(12.13, ") (d) OTHER INFORMATION: inote= This is a formacetal linkage." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:38:

GGTT G G T G T G GT T G G 5

(2) INFORMATION FOR SEQID NO:39: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (d) TOPOLOGY: linear ( i : ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(15, "") { D ) OTHER INFORMATION: Enote= This position indicates an abasic residue." ( x i y SEQUENCE DESCRIPTION: SEQID NO:39:

GGTT G G T G T G GTT GN 1 5

( 2) INFORMATION FOR SEQID No:40: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: mucleic acid 5,756.291 159 160 -continued ( C ) STRANDEDNESS; single ( D TOPOLOGY: linear ( i x ) FEATURE; ( A NAME/KEY: Inisc difference By LOCATION: replace(14." ( D OTHER INFORMATION: inote= This position is anabasic

( x i SEQUENCE DESCRIPTION: SEQID NO:40:

G T G G T G T G GT TNG is

( 2 INFORMATION FOR SEQID NO:41: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i : ) FEATURE; (ANAMEKEY: Inisc difference (B) LOCATION: replace(13,") ( D ) OTHER INFORMATION: note= This position is an abasic

( xi ) SEQUENCE DESCRIPTION: SEQID NO:41: G G T G G T G T G GTNG G is

( 2 INFORMATION FOR SEQID NO:42: { i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS; single (D) TOPOLOGY: linear ( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(12,") { D ) OTHER INFORMATION: /note= "This position is an abasic residue."

( i ) SEQUENCE DESCRIPTION: SEQED NO:42:

GGTT G G T G T G CNT is G 5

( 2) INFORMATION FOR SEQID NO:43: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE; (A) NAMEKEY: misc difference (B) LOCATION: replace(11,") (D) OTHER INFORMATION: Enote= This position is an abasic residue."

( x i SEQUENCE DESCRIPTION: SEQID No:43:

GT T G T G T G NT G. G. S

2 INFORMATION FOR SEQID NO:44: ( i y SEQUENCE CHARACTERISTICS: (ALENGTH: 15 base pairs ( B. TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear i x ) FEATURE: 5,756.291 161 162 -continued (A) NAMEKEY: misc difference (B) LOCATION: replace(10,") ( D.) OTHER INFORMATION: note= "This position is an abasic residue." ( xi ) SEQUENCE DESCRIPTION: SEQID No:44:

G GT T G G T G T N G T G G 5

( 2) INFORMATION FOR SEQID NO:45: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 15 base pairs ( B. ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference ( by LOCATION: replace09, "y ( D ) OTHER INFORMATION:/note= "This position is an abasic residue." ( x i ). SEQUENCE DESCRIPTION: SEQID NO:45:

G GT T G G T GNG G T G G 5

( 2) INFORMATION FOR SEQID NO:46: { i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(8,") ( D.) OTHER INFORMATION:/note "This position is an abasic residue." ( x i ). SEQUENCE DESCRIPTION: SEQID No:46:

GGTT G G T N T G GT TGG 1 5

( 2 INFORMATION FOR SEQID No:47: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE; mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE: (A) NAMB/KEY: misc difference (B) LOCATION: replace07. ") D.) OTHER INFORMATION: note "This position is an abasic residue." ( x i ) SEQUENCE DESCRIPTION: SEQED NO:47:

G G T T G GNG T G GT T G G 15

( 2) INFORMATION FOR SEQId No:48: ( i ) SEQUENCE CHARACTERISTICS: (A LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference ( B, ) LOCATION: replace(6,") (d) OTHER INFORMATION: note= "This position is an abasic residue." 5,756.291 163 164 -continued

( x i SEQUENCE DESCRIPTION: SEQID NO:48: GG T GN T G T G T G G 1 is

( 2) INFORMATION FOR SEQID NO:49: { i SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs ( B TYPE: nucleic acid C STRANDEDNESS: single ( D TOPOLOGY: linear ( 1 x ) FEATURE: ( A NAME/KEY: misc difference ( B LOCATION: replace(5. ") ( D OTHER INFORMATION: tnote= This position is an abasic residue." ( x i SEQUENCE DESCRIPTION: SEQD NO:49:

GGTT N G T G T G T G. 1 5

( 2 INFORMATION FOR SEQID No:50: i SEQUENCE CHARACTERISTICS: ( A y LENGTH: 15 base pairs (By TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( i x FEATURE: ( A NAMEKEY: Inisc difference ( B y LOCATION: replace(4.") ( D OTHER INFORMATION: note= This position is an abasic

( x i SEQUENCE DESCRIPTION: SEQED NO:50:

G G N G T G GT GG 15

( 2) INFORMATION FOR SEQID NO:51: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3,") { D ) OTHER INFORMATION: note= This position is an abasic residue." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:51:

GGNT G T G G T G 5

( 2 INFORMATION FOR SEQID NO:52: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(2,") ( D OTHER INFORMATION: inote= This is an abasic residue." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:52:

GNT T G G T G T G GTT G. G is 5,756.291 165 166 -continued

( 2) INFORMATION FOR SEQID NO:53: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(1,") (d) OTHER INFORMATION:/note= This is an abasic residue." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:53:

NG T G G T G G GT T G G

( 2 y. INFORMATION FOR SEQID NO:54: ( i ) SEQUENCE CHARACTERISTICs: ( A LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single { D TOPOLOGY: linear ( i x ) FEATURE: ( A y NAMBIKEY: misc difference ( B, ) LOCATION: replace(13, "). ( D ) OTHER INFORMATION: hote= This position is a 5-propynyl-2'-deoxyuridine residue." ( xi ) SEQUENCE DESCRIPTION: SEQID NO:54:

G G T T G G T G T G G T N G G

( 2) INFORMATION FOR SEQID NO:55: ( i ). SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: mucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace(12,") ( D ) OTHER INFORMATION: note= "This is a 5-propynyl-2'-deoxyuridine residue." ( x i ). SEQUENCE DESCRIPTION: SEQID NO:55:

GGTT G G T G T G GNT GG

( 2) INFORMATION FOR SEQID NO:56: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(9,") ( D OTHER INFORMATION: note= "This position is a 5-propynyl-2'-deoxyuridine residue." ( x i y SEQUENCE DESCRIPTION: SEQID No:56: GGTT G G T GNG GT T G G

( 2) INFORMATION FOR SEQID No:57: ( i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 15 base pairs 5,756,291 167 168 -continued (B) TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( i : ) FEATURE: (A) NAME/KEY: misc difference ( B, ) LOCATION: replace07, ") ( D.) OTHER INFORMATION: inote= This position is a 5-propynyl-2'-deoxyuridine residue.” ( x i y SEQUENCE DESCRIPTION: SEQED NO:57:

G. GT T G N G GT GG 5

( 2) INFORMATION FOR SEQID NO:58: ( i y SEQUENCE CHARACTERISTICS: ( A LENGE: 15 base pairs ( B. YPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear i : ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(4,") ( D.) OTHER INFORMATION: note= This position is a 5-propynyl-2'-deoxyuridine residue." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:58:

GGTNG G T G T G T G G 15

( 2 INFORMATION FOR SEQID NO:59: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid CSTRANDEDNESS: single D.) TOPOLOGY: linear ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3,") ( D OTHER INFORMATION: note "This position is a 5-propynyl-2'-deoxyuridine residue.” ( x i SEQUENCE DESCRIPTION: SEQID NO:59:

GNT G T G T G G T G G 1 is

( 2 INFORMATION FOR SEQID NO:60: ( ; ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B TYPE: nucleic acid ( C ), STRANDEDNESS: single ( D TOPOLOGY: linear ( i : ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(22, ") (D) OTHER INFORMATION: note= This is a 20 nucleotide stretch of randon sequences." ( x i ) SEQUENCE DESCRIPTION: SEQID NO:60: TAGAAT ACT C AAG CTTCGA C GNA GT T G GA. T C C C CGGGTA C 4

( 2 INFORMATION FOR SEQID NO:61: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single D TOPOLOGY: linear 5,756.291 169 170 -continued ( x i y SEQUENCE DESCRIPTION: SEQID NO:61:

TAGAAT ACT C A A G C T C G A C G 2

( 2) INFORMATION FOR SEQID No:62: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:62:

GTA C C C G GGG AT C CAAA CT 1 9

( 2) INFORMATION FOR SEQID NO:63: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid ( Cy STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:63:

A GTA T G T AT TA G T G TAG 9

(2) INFORMATION FOR SEQID NO:64: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:64:

A TAGA GTATA TAT G C T G T CT 2 O

( 2) INFORMATION FOR SEQID No:65: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No.65:

GTATATA GTA TA GTATTGGC 2 0.

( 2) INFORMATION FOR SEQID No.66: ( i) SEQUENCE CHARACTERISTICs: (A) LENGTH: 21 base pairs B ) TYPE: nucleic acid ( C ) STRANDEDNESS: single (d) TOPOLOGY: inear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:66:

A G GATA TAT G ATA T GATT C G G 2 1

( 2 INFORMATION FOR SEQID NO:67: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No.67: 5,756,291 171 172 -continued

A CTA CAT G AT ATA C C C

( 2) INFORMATION FOR SEQID NO:68: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs ( B, ) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:68:

CAT AAA C C C G A G CT. T. G 2 O

( 2) INFORMATION FOR SEQID NO-69: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS; single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQED No.69;

C C C CA AA C C C T A G C C 2

(2) INFORMATION FOR SEQID NO:70: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear x i ) SEQUENCE DESCRIPTION: SEQID No.70:

A C GCA C C G T A C C C C GT 7

( 2) INFORMATION FOR SEQID NO:71: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: Einear ( x i ) SEQUENCE DESCRIPTION: SEQID No.:71:

CA C CAAA CGC A T G CAT C C 2 0.

( 2 INFORMATION FOR SEQID NO:72: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs B TYPE: nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( i SEQUENCE DESCRIPTION: SEQID NO:72:

GTA CATT CA. G. G. C. G. C. CTG (CC 2 O

( 2 INFORMATION FOR SEQID No.:73: ( i SEQUENCE CHARACTERISTICS: (A ENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:73:

ACCA. T C C C G T GA CAA C 2 O 5,756.291 173 174 -continued

( 2) INFORMATION FOR SEQID NO:74: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No.:74:

GA CT AAA CGC AT T G T G C C C C 2 O

( 2) INFORMATION FOR SEQID No.75: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:75:

AAC GAA. GGG C A C GCC G G CT G 2 0

( 2) INFORMATION FOR SEQID No.:76: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No.:76: A CGG AT G. GT C T G GCT GG ACA 20

( 2) INFORMATION FOR SEQID No.:77: (i) SEQUENCE CHARACTERISTICS: ( A LENGTH: 60 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single ( D ) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No.:77: CGGGGA GAG G T G G T G G GT TGG CAA T G GC TAGA GTA GT G A CGTTTT C GC GGT CAC GT C C 6 O

( 2) INFORMATION FOR SEQID NO:78: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i y SEQUENCE DESCRIPTION: SEQID No.:78:

G G T G G G C G G T G G G T G G G 2 1

( 2 INFORMATION FOR SEQID NO:79: ( i SEQUENCE CHARACTERISTICs: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i x ) FEATURE: (ANAMEKEY: misc difference ( B LOCATION: replace(13.15, "") ( D OTHER INFORMATION: note= These positions are linked by thioate internucleotide linkages." 5,756.291 175 176 -continued ( x i ) SEQUENCE DESCRIPTION: SEQED NO:79:

G G T G G T G T G GTT G

( 2) INFORMATION FOR SEQID NO:80: ( i y SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( i : ) FEATURE: (A) NAMEKEY: misc difference ( B, ) LOCATION: replace(2,") ( D.) OTHER INFORMATION: inote "This position is pentynyl U.

{ i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(4,") (D) OTHER INFORMATION: note= This position is pentynyl U.

( i x ) FEATURE: (A) NAMEKEY: misc difference B ) LOCATION: replace06, ") ( D.) OTHER INFORMATION: note "This position is pentynyi U.'

( i : ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(9,") ( D.) OTHER INFORMATION: frote= This position is pentynyl du, { i x ) FEATURE: (A) NAME/KEY: misc difference ( B, ) LOCATION: replace(11,") ( D.) OTHER INFORMATION: fnote= "This position is peniyayi U. ( i x ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace014, ") D ) OTHER INFORMATION: note "This position is pentyny. U." ( i : ) FEATURE: (A) NAMEKEY: misc difference (B) LOCATION: replace16, ") (D) OTHER INFORMATION: note= This position is pentyny U.'

i x FEATURE: ( A NAMEKEY: misc difference ( B ) LOCATION: replace(17,") ( D.) OTHER INFORMATION: tnote= This position is peatynyl du." x i ) SEQUENCE DESCRIPTION: SEQID NO:80:

GNANANA GNA. NAGNANN C

( 2) INFORMATION FOR SEQID No:81: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: mucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear i r ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(3,") ( D.) OTHER INFORMATION: note= "his position is peatynyl ." 5,756,291 177 178 -continued ( i x ) FEATURE: (A) NAME/KEY: misc difference (B) LOCATION: replace(5,") (D) OIHER INFORMATION: note= "This position is pentynyl

( i x ) FEATURE: (A NAME/KEY: misc difference (B) LOCATION: replace09, ") ( D Y OTHER INFORMATION: /note= This position is pentynyl d."

( 1 x ) FEATURE: (A) NAME/KEY: misc difference B ) LOCATION: replace(11, ") { D ) OTHER INFORMATION: note "This position is pentynyl

( i x ) FEATURE: (A) NAME/KEY: misc difference ( By LOCATION: replace012, "") (D) OTHER INFORMATION: note= This position is penrynyl

( i x FEATURE: (A) NAMEKEY: misc-difference (B) LOCATION: replace(15,") D.) OTHER INFORMATION:/note= "This position is penynyl

( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(17. ") (d) OHER INFORMATION: inote= This position is pentynyi

( i x ) FEATURE; (A) NAMEKEY: misc difference (B) LOCATION: replace(20,") (D) OTHER INFORMATION: tnote= "This position is pentynyl

( xi ) SEQUENCE DESCRIPTION: SEQD NO:81:

G CNG NAGANA NNAGNANAGN 2 0.

( 2) INFORMATION FOR SEQID No:82: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: mucleic acid (CSTRANDEDNESS: single ( D TOPOLOGY: linear ( i x ) FEATURE; (A) NAME/KEY: misc difference (B) LOCATION: replace(20,") (d) OTHER INFORMATION: note= "This is a 60 nucleotide stretch of random sequences." ( xi ) SEQUENCE DESCRIPTION: SEQID No.82: TGG CAGA T G A C C C GAATT C N G GAT C CAG T G CT CAAA T G T 4 0

( 2 INFORMATION FOR SEQID NO:83: ( i SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 19 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:83:

T G. G. CAGAT GA C C C GAATTC 19

( 2) INFORMATION FOR SEQID No:84: 5,756,291 179 180 -continued

( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 20 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear x i y SEQUENCE DESCRIPTION: SEQID NO:84:

AA CAT GAG CA CTC GATCC

2 ) INFORMATION FOR SEQID No.85: ( ; ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 18 amino acids (B) TYPE:amino acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQ ID NO:85: Ty r A s p G in P r c L y s Le u Le u G y I 1 e G i u Thr Pro Le u Pro Lys Lys 5 O 5

G u Le u

( 2) INFORMATION FOR SEQID NO:36: ( i ). SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i ). SEQUENCE DESCRIPTION: SEQID NO:86: Thr Pro Le u Pro Lys Lys G 1 u, Le u L. eu Le u Pro G 1 y As a A is in A rig , y is 5 O 5 Wa 1 Tyr G 1 u

( 2) INFORMATION FOR SEQID NO:87: ( i y SEQUENCE CHARACTERISTICs: ( A LENGTH: 19 amino acids ( B TYPE: amino acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID NO:87: Le u Pro G y As n. As in Arg Lys v a 1 Tyr G u Le u se r A s in W a 1 G in G 1 u 5 O 15

A s p Ser G 1 a

( 2) INFORMATION FOR SEQID NO:88: ( i SEQUENCE CHARACTERISTICS: (A) LENGTE: 18 annino acids (B) TYPE: amino acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID NO:88: Ty r S e r A is in Cys Pro A s p G i y G 1 in Ser Th r A a Lys Thr P he Le u T h r 1 5 0 15 w a 1 Tyr

( 2) INFORMATION FOR SEQID NO:89: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids 5,756,291 181 182 -continued (B) TYPE:amino acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:89: Ty r G i u Le u A s p Le u A rig P T o G l n G 1 y Le u G 1 u Le u Ph e G i u As n Thr 1. 5 O 5

S e r

( 2 INFORMATION FOR SEQID No.90: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 base pairs B ) TYPE; nucleic acid ( C ), STRANDEDNESS: single (D) TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:90:

GGGTT G G G T C G G T G GT 7

( 2) INFORMATION FOR SEQID No.91: ( i ) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid Cy STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQED NO:91:

GG GAT GGTTT G GT T G GG 1 7

( 2 INFORMATION FOR SEQID No.92: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: mucleic acid ( C ) STRANDEDNESS: single (d) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO92:

AG G T T G G GAG G G T G G G 1 6

( 2) INFORMATION FOR SEQID No:93: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (d) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:93:

TGG G GC GA. GG AT G GA 7

( 2) INFORMATION FOR SEQID NO:94: ( i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (d) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:94: AGGTTGG GTA GT GT T G GT 18

( 2) INFORMATION FOR SEQID NO:95: ( ; ) SEQUENCE CHARACTERISTICS: 5,756.291 183 184 -continued ( A LENGTH: 17 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single (D) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO95:

AG G T G G G C G G T G - G 7

( 2) INFORMATION FOR SEQID NO:96: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 16 base pairs (B) TYPE: nucleic acid C ) STRANDEDNESS: single ( D.) TOPOLOGY: linear ( x i ) SEQUENCE DESCRIPTION: SEQID NO:96:

GGG T G G GA G G T G GA 1 5

( 2 INFORMATION FOR SEQED NO:97: ( i ) SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 base pairs ( B TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( xi ) SEQUENCE DESCRIPTION: SEQID No.97: T G T G G C G G T G GG 7

( 2 INFORMATION FOR SEQID NO:98: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (By TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQED NO:98: GA G G T G T G T G GC

( 2) INFORMATION FOR SEQID No.99: ( i SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS; single (D) TOPOLOGY: Einear ( x i y SEQUENCE DESCRIPTION: SEQID NO99:

T G G CAG GA. GGG 1 7

( 2 INFORMATION FOR SEQ ID No:100: ( i SEQUENCE CHARACTERISTICS: ( A LENGTH: 17 base pairs (B) TYPE: nucleic acid ( C STRANDEDNESS: single ( D TOPOLOGY: linear ( x i SEQUENCE DESCRIPTION: SEQID No:100:

G GAT G G T G A G G T G GA 7

( 2 INFORMATION FOR SEQID No:101: { i ). SEQUENCE CHARACTERISTICS: (A LENGTH: 17 base pairs (B) TYPE: mucleic acid