AU725590B2 - High affinity nucleic acid ligands to lectins - Google Patents

Description

WO 96/40703 PCT/US96/09455 High Affinity Nucleic Acid Ligands to Lectins FIELD OF THE INVENTION Described herein are methods for identifying and preparing high-affinity nucleic acid ligands to lectins. Lectins are carbohydrate binding proteins. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential enrichment.

Specifically disclosed herein are high-affinity nucleic acid ligands to wheat germ agglutinin (WGA), L-selectin, E-selectin, and P-selectin.

BACKGROUND OF THE INVENTION The biological role of lectins (non-enzymatic carbohydrate-binding proteins of non-immune origin; I. J. Goldstein et al., 1980, Nature 285:66) is inextricably linked to that of carbohydrates. One function of carbohydrates is the modification of physical characteristics of glyco-conjugates solubility, stability, activity, susceptibility to enzyme or antibody recognition), however, a more interesting and relevant aspect of carbohydrate biology has emerged in recent years; the carbohydrate portions of glyco-conjugates are information rich molecules (N.

Sharon and H. Lis, 1989, Science 246:227-234; K. Drickamer and M. Taylor, 1993, Annu. Rev. Cell Biol. 9:237-264; A. Varki, 1993, Glycobiol. 3:97-130).

Within limits, the binding of carbohydrates by lectins is specific there are lectins that bind only galactose or N-acetylgalactose; other lectins bind mannose; still others bind sialic acid and so on; K. Drickamer and M. Taylor, supra). Specificity of binding enables lectins to decode information contained in the carbohydrate portion of glyco-conjugates and thereby mediate many important biological functions.

Numerous mammalian, plant, microbial and viral lectins have been described Ofek and N. Sharon, 1990, Current Topics in Microbiol.and Immunol. 151:91- 113; K. Drickamer and M. Taylor, supra; I. J. Goldstein and R. D. Poretz, 1986, in The Lectins, p.p. 33-247; A. Varki, supra). These proteins mediate a diverse array of biological processes which include: trafficking of lysosomal enzymes, clearance of serum proteins, endocytosis, phagocytosis, opsonization, microbial and viral infections, toxin binding, fertilization, immune and inflammatory responses, cell adhesion and migration in development and in pathological conditions such as metastasis. Roles in symbiosis and host defense have been proposed for plant WO 96/40703 PCT/US96/09455 2 lectins but remain controversial. While the functional role of some lectins is well understood, that of many others is understood poorly or not at all.

The diversity and importance of processes mediated by lectins is illustrated by two well documented mammalian lectins, the asialoglycoprotein receptor and the serum mannose binding protein, and by the viral lectin, influenza virus hemagglutinin. The hepatic asialoglycoprotein receptor specifically binds galactose and N-acetylgalactose and thereby mediates the clearance of serum glycoproteins that present terminal N-acetylgalactose or galactose residues, exposed by the prior removal of a terminal sialic acid. The human mannose-binding protein (MBP) is a serum protein that binds terminal mannose, fucose and N-acetylglucosamine residues. These terminal residues are common on microbes but not mammalian glyco-conjugates. The binding specificity of MBP constitutes a non-immune mechanism for distinguishing self from non-self and mediates host defense through opsonization and complement fixation. Influenza virus hemagglutinin mediates the initial step of infection, attachment to nasal epithelial cells, by binding sialic acid residues of cell-surface receptors.

The diversity of lectin mediated functions provides a vast array of potential therapeutic targets for lectin antagonists. Both lectins that bind endogenous carbohydrates and those that bind exogenous carbohydrates are target candidates.

For example, antagonists to the mammalian selectins, a family of endogenous carbohydrate binding lectins, may have therapeutic applications in a variety of leukocyte-mediated disease states. Inhibition of selectin binding to its receptor blocks cellular adhesion and consequently may be useful in treating inflammation, coagulation, transplant rejection, tumor metastasis, rheumatoid arthritis, reperfusion injury, stroke, myocardial infarction, burs, psoriasis, multiple sclerosis, bacterial sepsis, hypovolaemic and traumatic shock, acute lung injury, and ARDS.

The selectins, P- and are three homologous C-type lectins that recognize the tetrasaccharide, sialyl-Lewis x Foxall et al, 1992, J. Cell Biol.

117,895-902). Selectins mediate the initial adhesion of neutrophils and monocytes to activated vascular endothelium at sites of inflammation S. Cotran et al., 1986, J. Exp. Med. 164, 661-; M. A. Jutila et al., 1989, J. Immunol. 143,3318-; J. G.

Geng et al., 1990, Nature, 757; U. H. Von Adrian et al., 1994, Am. J. Physiol.

Heart Circ. Physiol. 263, H1034-H1044). In addition, L-selectin is responsible for the homing of lymphocytes to peripheral and mesenteric lymph nodes M.

Gallatin et al., 1983, Nature 304,30; T. K. Kishimoto et al., 1990, Proc. Natl.

Acad. Sci. 87,2244-) and P-selectin mediates the adherence of platelets to neutrophils and monocytes Hsu-Lin et al., 1984, J. BioL Chem. 259,9121).

WO 96/40703 PCT/US96/09455 3 Selectin antagonists (antibodies and carbohydrates) have been shown to block the extravasation of neutrophils at sites of inflammation Piscueta and F.

W. Luscinskas, 1994, Am. J. Pathol. 145,461-469), to be efficacious in animal models of ischemia/reperfusion Weyrich et al., 1993, J. Clin. Invest.

91,2620-2629; R.K. Winn et al., 1993, J. Clin. Invest. 92, 2042-2047), acute lung injury Mulligan et al., 1993, J. Immunol. 151, 6410-6417; A. Seekamp et al., 1994, Am. J. Pathol. 144, 592-598), insulitis/diabetes Yang et al., 1993, Proc. Natl. Acad. Sci. 90,10494-10498), meningitis Granet et al., 1994, J.

Clin. Invest. 93, 929-936), hemorrhagic shock Winn et al., 1994, Am J.

Physiol. Heart Circ. Physiol. 267, H2391-H2397) and transplantation. In addition, selectin expression has been documented in models of arthritis Jamar et al., 1995, Radiology 194, 843-850), experimental allergic encephalomyelitis

(J.M.

Dopp et al., 1994, J. Neuroimmunol. 54, 129-144), cutaneous inflammation (A.

Siber et al., 1994, Lab. Invest. 70, 163-170) glomerulonephritis Tipping et al., 1994, Kidney Int. 46,79-88), on leukaemic cells and colon carcinomas (R.M.

Lafrenie et al., 1994, Eur. J. Cancer 30A, 2151-2158) and L-selectin receptors have been observed in myelinated regions of the central nervous system Huang et al., 1991, J. Clin. Invest. 88, 1778-1783). These animal model data strongly support the expectation of a therapeutic role for selectin antagonists in a wide variety of disease states in which host tissue damage is neutrophil-mediated.

Other examples of lectins that recognize endogenous carbohydrates are CD22p, CD23, CD44 and sperm lectins Varki, 1993, Glycobiol.3, 97-130; P.M. Wassarman, 1988, Ann. Rev. Biochem. 57, 415-442). CD223 is involved in early stages of B lymphocyte activation; antagonists may modulate the immune response. CD23 is the low affinity IgE receptor; antagonists may modulate the IgE response in allergies and asthma. CD44 binds hyaluronic acid and thereby mediates cell/cell and cell/matrix adhesion; antagonists may modulate the inflammatory response. Sperm lectins are thought to be involved in sperm/egg adhesion and in the acrosomal response; antagonists may be effective contraceptives, either by blocking adhesion or by inducing a premature, spermicidal acrosomal response.

Antagonists to lectins that recognize exogenous carbohydrates may have wide application for the prevention of infectious diseases. Many viruses (influenza A, B and C; Sendhi, Newcastle disease, coronavirus, rotavirus, encephalomyelitis virus, enchephalomyocarditis virus, reovirus, paramyxovirus) use lectins on the surface of the viral particle for attachment to cells, a prerequisite for infection; antagonists to these lectins are expected to prevent infection (A.Varki, 1993, Glycobiol.3, 97-130). Similarly colonization/infection strategies of many bacteria WO 96/40703 PCT/US96/09455 4 utilize cell surface lectins to adhere to mammalian cell surface glyco-conjugates.

Antagonists to bacterial cell surface lectins are expected to have therapeutic potential for a wide spectrum of bacterial infections, including: gastric (Helicobacter pylori), urinary tract coli), pulmonary (Klebsiella pneumoniae, Stretococcus pneumoniae, Mycoplasma pneumoniae) and oral (Actinomyces naeslundi and Actinomyces viscosus) colonization/infection Abraham, 1994, Bacterial Adhesins, in The Handbook of Immunopharmacology: Adhesion Molecules, C.D.

Wegner, ed; B.J. Mann et al., 1991, Proc. Natl. Acad. Sci. 88, 3248-3252). A specific bacterial mediated disease state is Pseudomonas aeruginosa infection, the leading cause of morbidity and mortality in cystic fibrosis patients. The expectation that high affinity antagonists will have efficacy in treating P. aeruginosa infection is based on three observations. First, a bacterial cell surface, GalNAcl1-4Gal binding lectin mediates infection by adherence to asialogangliosides (aGM1 and aGM2) of pulmonary epithelium Imundo et al., 1995, Proc. Natl. Acad. Sci 92, 3019-3023). Second, in vitro, the binding of P. aeruginosa is competed by the gangliosides' tetrasaccharide moiety, Galpl-3GalNAcl-4Gall-4Glc. Third, in vivo, instillation of antibodies to Pseudomonas surface antigens can prevent lung and pleural damage Pittet et al., 1993, J. Clin. Invest. 92, 1221-1228).

Non-bacterial microbes that utilize lectins to initiate infection include Entamoeba histalytica (a Gal specific lectin that mediates adhesion to intestinal mucosa; W.A. Petri, Jr., 1991, AMS News 57:299-306) and Plasmodium faciparum (a lectin specific for the terminal Neu5Ac(a2-3)Gal of glycophorin A of erthrocytes; P.A. Orlandi et al., 1992, J. Cell Biol. 116:901-909). Antagonists to these lectins are potential therapeutics for dysentery and malaria.

Toxins are another class of proteins that recognize exogenous carbohydrates (K-A Karlsson, 1989, Ann. Rev. Biochem. 58:309-350). Toxins are complex, two domain molecules, composed of a functional and a cell recognition/adhesion domain. The adhesion domain is often a lectin bacterial toxins: pertussis toxin, cholera toxin, heat labile toxin, verotoxin and tetanus toxin; plant toxins: ricin and abrin). Lectin antagonists are expected to prevent these toxins from binding their target cells and consequently to be useful as antitoxins.

There are still other conditions for which the role of lectins is currently speculative. For example, genetic mutations result in reduced levels of the serum mannose-binding protein (MBP). Infants who have insufficient levels of this lectin suffer from severe infections, but adults do not. The high frequency of mutations in both oriental and Caucasian populations suggests a condition may exist in which low levels of serum mannose-binding protein are advantageous. Rheumatoid arthritis WO 96/40703 PCT/US96/09455 (RA) may be such a condition. The severity of RA is correlated with an increase in IgG antibodies lacking terminal galactose residues on Fc region carbohydrates (A.

Young et al., 1991, Arth. Rheum. 34, 1425-1429; I.M. Roitt et al., 1988, J.

Autoimm. 1,499-506). Unlike their normal counterpart, these gal-deficient carbohydrates are substrates for MBP. MBP/IgG immunocomplexes may contribute to host tissue damage through complement activation. Similarly, the eosinophil basic protein is cytotoxic. If the cytotoxicity is mediated by the lectin activity of this protein, then a lectin antagonist may have therapeutic applications in treating eosinophil mediated lung damage.

Lectin antagonists may also be useful as imaging agents or diagnostics. For example, E-selectin antagonists may be used to image inflamed endothelium.

Similarly antagonists to specific serum lectins, i.e. mannose-binding protein, may also be useful in quantitating protein levels.

Lectins are often complex, multi-domain, multimeric proteins. However, the carbohydrate-binding activity of mammalian lectins is normally the property of a carbohydrate recognition domain or CRD. The CRDs of mammalian lectins fall into three phylogenetically conserved classes: C-type, S-type and P-type Drickamer and M.E. Taylor, 1993, Annu. Rev. Cell Biol. 9, 237-264). C-type lectins require Ca for ligand binding, are extracellular membrane and soluble proteins and, as a class, bind a variety of carbohydrates. S-type lectins are most active under reducing conditions, occur both intra- and extracellularly, bind (-galactosides and do not require P-type lectins bind mannose 6-phosphate as their primary ligand.

Although lectin specificity is usually expressed in terms of monosaccharides and/or oligosacchrides MBP binds mannose, fucose and Nacetylglucosamine), the affinity for monosaccharides is weak. The dissociation constants for monomeric saccharides are typically in the millimolar range Lee, 1992, FASEB J. 6:3193-3200; G.D. Glick et al., 1991, J Biol.Chem. 266:23660- 23669; Y. Nagata and M.M. Burger, 1974, J. Biol. Chem. 249:116-3122).

Co-crystals of MBP complexed with mannose oligomers offer insight into the molecular limitations on affinity and specificity of C-type lectins Weis et al., 1992, Nature 360:127-134; K. Drickamer, 1993, Biochem. Soc. Trans. 21:456- 459). The 3- and 4-hydroxyl groups of mannose form coordination bonds with bound Ca++ ion #2 and hydrogen bonds with glutamic acid (185 and 193) and asparagine (187 and 206). The limited contacts between the CRD and bound sugar are consistent with its spectrum of monosaccharide binding; N-acetylglucosamine has equatorial 3- and 4-hydroxyls while fucose has similarly configured hydroxyls at the 2 and 3 positions.

WO 96/40703 PCT/US96/09455 6 The affinity of the mannose-binding protein and other lectins for their natural ligands is greater than that for monosaccharides. Increased specificity and affinity can be accomplished by establishing additional contacts between a protein and its ligand Drickamer, 1993, supra) either by 1) additional contacts with the terminal sugar chicken hepatic lectin binds N-acetylglucose amine with greater affinity than mannose or fucose suggesting interaction with the 2-substituent); 2) clustering of CRDs for binding complex oligosaccharides the mammalian asialylglycoprotein receptor); 3) interactions with additional saccharide residues the lectin domain of selectins appears to interact with two residues of the tetrasaccharide sialyl-LewisX: with the charged terminal residue, sialic acid, and with the fucose residue; wheat germ agglutinin appears to interact with all three residues of trimers of N-acetylglucosamine); or by 4) contacts with a noncarbohydrate portion of a glyco-protein.

The low affinity of lectins for mono- and oligo-saccharides presents major difficulties in developing high affinity antagonists that may be useful therapeutics.

Approaches that have been used to develop antagonists are similar to those that occur in nature: 1) addition or modification of substituents to increase the number of interactions; and 2) multimerization of simple ligands.

The first approach has had limited success. For example, homologues of sialic acid have been analyzed for affinity to influenza virus hemagglutinin

(S.J.

Watowich et al. 1994, Structure 2:719-731). The dissociation constants of the best analogues are 30 to 300 pM which is only 10 to 100-fold better than the standard monosaccharide.

Modifications of carbohydrate ligands to the selectins have also had limited success. In static ELISA competition assays, sialyl-Lewisa and sialyl-Lewisx have IC50s of 220 pM and 750 iM, respectively, for the inhibition of the binding of an E-selectin/IgG chimera to immobilized sialyl-Lewisx Nelson et al., 1993, J.

Clin. Invest. 91, 1157-1166). The IC50 of a sialyl-Lewisa derivative (addition of an aliphatic aglycone to the GlcNAc and replacement of the N-acetyl with an NH2 group) improved 10-fold to 21 pM. Similarly, removal of the N-acetyl from sialyllewis x improves inhibition in an assay dependent manner Foxall et al., 1992, J.

Cell Biol. 117, 895-902; S.A. DeFrees et al., 1993, J. Am. Chem. Soc. 115, 7549- 7550).

The second approach, multimerization of simple ligands, can lead to dramatic improvements in affinity for lectins that bind complex carbohydrates Lee, supra). On the other hand, the approach does not show great enhancement for lectins that bind simple oligosaccharides; dimerizing sialyl-Lewisx, a minimal WO 96/40703 PCT/US96/09455 7 carbohydrate ligand for E-selectin, improves inhibition approximately 5-fold (S.A.

DeFrees et al., supra).

An alternative approach is to design compounds that are chemically unrelated to the natural ligand. In the static ELISA competition assays inositol polyanions inhibit L- and P-selectin binding with IC50s that range from 1.4 PM to 2.8 mM (0.

Cecconi et al., 1994, J. Biol. Chem. 269, 15060-15066). Synthetic oligopeptides, based on selectin amino acid sequences, inhibit neutrophil binding to immobilized Pselectin with IC50s ranging from 50 pM to 1 mM (J-G Geng et al., 1992, J of Biol.

Chem. 267, 19846-19853).

Lectins are nearly ideal targets for isolation of antagonists by SELEX technology described below. The reason is that oligonucleotide ligands that are bound to the carbohydrate binding site can be specifically eluted with the relevant sugar(s). Oligonucleotide ligands with affinities that are several orders of magnitude greater than that of the competing sugar can be obtained by the appropriate manipulation of the nucleic acid ligand to competitor ratio. Since the carbohydrate binding site is the active site of a lectin, essentially all ligands isolated by this procedure will be antagonists. In addition, these SELEX ligands will exhibit much greater specificity than monomeric and oligomeric saccharides.

A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in United States Patent Application Serial No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled "Nucleic Acid Ligands," now United States Patent Number 5,475,096, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled "Nucleic Acid Ligands," now United States Patent No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.

The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, WO 96/40703 PCT/US96/09455 8 partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.

The basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No. 07/960,093, filed October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure," describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. United States Patent Application Serial No. 08/123,935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule.

United States Patent Application Serial No. 08/134,028, filed October 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. United States Patent Application Serial No. 07/964,624, filed October 21, 1992, entitled "Methods of Producing Nucleic Acid Ligands" describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. United States Patent Application Serial No. 08/400,440, filed March 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," describes methods for covalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.

Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in United States Patent Application Serial No.

08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," that describes oligonucleotides containing WO 96/40703 PCT/US96/09455 9 nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines.

United States Patent Application Serial No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'amino 2'-fluoro and/or 2'-O-methyl United States Patent Application Serial No. 08/264,029, filed June 22, 1994, entitled "Novel Method of Preparation of 2' Modified Pyrimidine Intramolecular Nucleophilic Displacement," describes novel methods for making 2-modified nucleosides.

The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX". The SELEX method also includes combining the selected nucleic acid ligands with non-oligonucleotide functional units and United States Patent Application Serial No. 08/234,997, filed April 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX" and United States Patent Application Serial No.

08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes". These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.

The present invention applies the SELEX methodology to obtain nucleic acid ligands to lectin targets. Lectin targets, or lectins, include all the non-enzymatic carbohydrate-binding proteins of non-immune origin, which include, but are not limited to, those described above.

Specifically, high affinity nucleic acid ligands to wheat germ agglutinin, and various selectin proteins have been isolated. For the purposes of the invention the terms wheat germ agglutinin, wheat germ lectin and WGA are used interchangeably.

Wheat germ agglutinin (WGA) is widely used for isolation, purification and structural studies of glyco-conjugates. As outlined above, the selectins are important anti-inflammatory targets. Antagonists to the selectins modulate extravasion of leukocytes at sites of inflammation and thereby reduce neutrophil caused host tissue damage. Using the SELEX technology, high affinity antagonists of L-selectin, Eselectin and P-selectin mediated adhesion are isolated.

WO 96/40703 PCT/US96/09455 BRIEF SUMMARY OF THE INVENTION The present invention includes methods of identifying and producing nucleic acid ligands to lectins and the nucleic acid ligands so identified and produced. More particularly, nucleic acid ligands are provided that are capable of binding specifically to Wheat Germ Agglutinin (WGA), L-Selectin, E-selectin and P-selectin.

Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to lectins comprising the steps of (a) preparing a candidate mixture of nucleic acids, partitioning between members of said candidate mixture on the basis of affinity to said lectin, and amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to said lectin.

More specifically, the present invention includes the nucleic acid ligands to lectins identified according to the above-described method, including those ligands to Wheat Germ Agglutinin listed in Table 2, those ligands to L-selectin listed in Tables 8, 12 and 16, and those ligands to P-selectin listed in Tables 19 and Additionally, nucleic acid ligands to E-selectin and serum mannose binding protein are provided. Also included are nucleic acid ligands to lectins that are substantially homologous to any of the given ligands and that have substantially the same ability to bind lectins and antagonize the ability of the lectin to bind carbohydrates. Further included in this invention are nucleic acid ligands to lectins that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind lectins and antagonize the ability of the lectin to bind carbohydrates.

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

The present invention also includes the use of the nucleic acid ligands in therapeutic, prophylactic and diagnostic applications.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows consensus hairpin secondary structures for WGA 2'-NH2 RNA ligands: family 1, family 2 and family 3. Nucleotide sequence is in standard one letter code. Invariant nucleotides are in bold type. Nucleotides derived from fixed sequence are in lower case.

Figure 2 shows binding curves for the L-selectin SELEX second and ninth round 2'-NH2 RNA pools to peripheral blood lymphocytes (PBMCs).

WO 96/40703 PCT/US96/09455 11 Figure 3 shows binding curves for random 40N7 2'-NH2 RNA (SEQ ID NO: 64) and the cloned L-selectin ligand, F14.12 (SEQ ID NO: 78), to peripheral blood lymphocytes (PBMC).

Figure 4 shows the results of a competition experiment in which the binding of 5 nM 3 2 p-labeled F14.12 (SEQ ID NO: 78) to PBMCs (10 7 /ml) is competed with increasing concentrations of unlabeled F14.12 (SEQ ID NO: 78). RNA Bound equals 100 x (net counts bound in the presence of competitor/net counts bound in the absence of competitor).

Figure 5 shows the results of a competition experiment in which the binding of 5 nM 32 p-labeled F14.12 (SEQ ID NO: 78) to PBMCs (107/ml) is competed with increasing concentrations of the blocking monoclonal anti-L-selectin antibody, DREG-56, or an isotype matched, negative control antibody. RNA Bound equals 100 x (net counts bound in the presence of competitor/net counts bound in the absence of competitor).

Figure 6 shows the results of a competitive ELISA assay in which the binding of soluble LS-Rg to immobilized sialyl-LewisX/BSA conjugates is competed with increasing concentrations of unlabeled F14.12 (SEQ ID NO: 78). Binding of LS-Rg was monitored with an HRP conjugated anti-human IgG antibody. LS-Rg Bound equals 100 x (OD450 in the presence of competitor)/(OD450 in the absence of competitor). The observed OD450 was corrected for nonspecific binding by subtracting the OD450 in the absence of LS-Rg from the experimental values. In the absence of competitor the OD450 was 0.324 and in the absence of LS-Rg 0.052.

Binding of LS-Rg requires divalent cations; in the absence of competitor, replacement of with 4 mM EDTA reduced the OD450 to 0.045.

Figure 7 shows hairpin secondary structures for representative L-selectin 2'NH2 RNA ligands: F13.32 (SEQ. ID NO: 67), family I; 6.16 (SEQ. ID NO: 84), family mI; and F14.12 (SEQ. ID NO: 78), family II. Nucleotide sequence is in standard one letter code. Invariant nucleotides are in bold type.

Nucleotides derived from fixed sequence are in lower case.

Figure 8 shows a schematic representation of each dimeric and mutimeric oligonucleotide complex: dimeric branched oligonucleotide; multivalent streptavidin/bio-oligonucleotide complex streptavidin; B: biotin); dimeric dumbell oligonucleotide; dimeric fork oligonucleotide.

Figure 9 shows binding curves for the L-selectin SELEX fifteenth round ssDNA pool to PBMCs (10 7 /ml).

Figure 10 shows the results of a competition experiment in which the binding of 2 nM 3 2 p-labeled round 15 ssDNA to PBMCs (10 7 /ml) is competed with WO 96/40703 PCT/US96/09455 12 increasing concentrations of the blocking monoclonal anti-L-selectin antibody, DREG-56, or an isotype matched, negative control antibody. RNA Bound equals 100 x (net counts bound in the presence of competitor/net counts bound in the absence of competitor).

Figure 11 shows L-selectin specific binding ofLD201T1 (SEQ ID NO: 185) to human lymphocytes and granulocytes in whole blood, a, FITC-LD201T1 binding to lymphocytes is competed by DREG-56, unlabeled LD201T1, and inhibited by EDTA. b, FITC-LD201T1 binding to granulocytes is competed by DREG-56, unlabeled LD201T1, and inhibited by EDTA. All samples were stained with 0.15 mM FITC-LD201T1; thick line: FITC-LD201T1 only; thick dashed line: FITC- LD201T1 with 0.3 mM DREG-56; medium thick line: FITC-LD201T1 with 7 mM unlabeled NX280; thin line: FITC-LD201T1 stained cells, reassayed after addition of 4 mM EDTA; thin dashed line: autofluorescence.

Figure 12 shows the consensus hairpin secondary structures for family 1 ssDNA ligands to L-selectin. Nucleotide sequence is in standard one letter code.

Invariant nucleotides are in bold type. The base pairs at highly variable positions are designated To the right of the stem is a matrix showing the number of occurances of particular base pairs for the position in the stem that is on the same line.

Figure 13 shows that in vitro pre-treatment of human PBMC with NX288 (SEQ ID NO: 193) inhibits lymphocyte trafficking to SCID mouse PLN. Human PBMC were purified from heparinised blood by a Ficoll-Hypaque gradient, washed twice with HBSS (calcium/magnesium free) and labeled with 5 1 Cr (Amersham).

After labeling, the cells were washed twice with HBSS (containing calcium and magnesium) and 1% bovine serum albumin (Sigma). Female SCID mice (6-12 weeks of age) were injected intravenously with 2x10 6 cells. The cells were either untreated or mixed with either 13 pmol of antibody (DREG-56 or MEL-14), or 4, 1, or 0.4 nmol of modified oligonucleotide. One hour later the animals were anaesthetised, a blood sample taken and the mice were euthanised. PLN, MLN, Peyer's patches, spleen, liver, lungs, thymus, kidneys and bone marrow were removed and the counts incorporated into the organs determined by a Packard gamma counter. Values shown represent the mean s.e. of triplicate samples, and are representative of 3 experiments.

Figure 14 shows that pre-injection of NX288 (SEQ ID NO: 193) inhibits human lymphocyte trafficking to SCID mouse PLN and MLN. Human PBMC were purified, labeled, and washed as described above. Cells were prepared as described in Figure 13. Female SCID mice (6-12 weeks of age) were injected intravenously WO 96/40703 PCT/US96/09455 13 with 2x10 6 cells. One to 5 min prior to injecting the cells, the animals were injected with either 15 pmol DREG-56 or 4 nmol modified oligonucleotide. Animals were scarificed 1 hour after injection of cells. Counts incorporated into organs were quantified as described in Figure 13. Values shown represent the mean s.e. of triplicate samples, and are representative of 2 experiments.

Figure 15 shows the consensus hairpin secondary structures for 2'-F RNA ligands to L-selectin. Nucleotide sequence is in standard one letter code. Invariant nucleotides are in bold type. The base pairs at highly variable positions are designated To the right of the stem is a matrix showing the number of occurances of particular base pairs for the position in the stem that is on the same line.

Figure 16 shows the consensus hairpin secondary structures for 2'-F RNA ligands to P-selectin. Nucleotide sequence is in standard one letter code. Invariant nucleotides are in bold type. The base pairs at highly variable positions are designated To the right of the stem is a matrix showing the number of occurances of particular base pairs for the position in the stem that is on the same line.

DETAILED DESCRIPTION OF THE INVENTION Thisapplication describes high-affinity nucleic acid ligands to lectins identified through the method known as SELEX. SELEX is described in U.S.

Patent Application Serial No. 07/536,428, entitled "Systematic Evolution of Ligands by EXponential Enrichment", now abandoned; U.S. Patent Application Serial No.

07/714,131, filed June 10, 1991, entitled "Nucleic Acid Ligands", now United States Patent No. 5,475,096; United States Patent Application Serial No.

07/931,473, filed August 17, 1992, entitled "Nucleic Acid Ligands", now United States Patent No. 5,270,163, (see also PCT/US91/04078). These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.

In its most basic form, the SELEX process may be defined by the following series of steps: 1) A candidate mixture of nucleic acids of differing sequence is prepared.

The candidate mixture generally includes regions of fixed sequences each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: to assist in the amplification steps described below, to mimic WO 96/40703 PCT/US96/09455 14 a sequence known to bind to the target, or to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized the probability of finding a base at any position being one in four) or only partially randomized the probability of finding a base at any location can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acidtarget pairs between the target and those nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately .05-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.

By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.

Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixture. The SELEX Patent Applications also describe ligands obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.

This invention also includes the ligands as described above, wherein certain chemical modifications are made in order to increase the in vivo stability of the ligand or to enhance or mediate the delivery of the ligand. Examples of such WO 96/40703 PCT/US96/09455 modifications include chemical substitutions at the sugar and/ or phosphate and/or base positions of a given nucleic acid sequence. See, U.S. Patent Application Serial No. 08/117,991, filed September 9, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides" which is specifically incorporated herein by reference. Additionally, in co-pending and commonly assigned U.S.

Patent Application Serial No. 07/964,624, filed October 21, 1992 now U.S.

Patent No. 5,496,938, methods are described for obtaining improved nucleic acid ligands after SELEX has been performed. The '624 application, entitled "Methods of Producing Nucleic Acid Ligands," is specifically incorporated herein by reference. Further included in the '624 patent are methods for determining the threedimensional structures of nucleic acid ligands. Such methods include mathematical modeling and structure modifications of the SELEX-derived ligands, such as chemical modification and nucleotide substitution. Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEX (modification of previously identified unmodified ligands) or by incorporation into the SELEX process. Additionally, the nucleic acid ligands of the invention can be complexed with various other compounds, including but not limited to, lipophilic compounds or non-immunogenic, high molecular weight compounds. Lipophilic compounds include, but are not limited to, cholesterol, dialkyl glycerol, and diacyl glycerol. Non-immunogenic, high molecular weight compounds include, but are not limited to, polyethylene glycol, dextran, albumin and magnetite. The nucleic acid ligands described herein can be complexed with a lipophilic compound cholesterol) or attached to or encapsulated in a complex comprised of lipophilic components a liposome). The complexed nucleic acid ligands can enhance the cellular uptake of the nucleic acid ligands by a cell for delivery of the nucleic acid ligands to an intracellular target. The complexed nucleic acid ligands can also have enhanced pharmacokinetics and stability. United States Patent Application Serial Number 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes," which is herein incorporated by reference describes a method for preparing a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipophilic compound or a non-immunogenic, high molecular weight compound.

The methods described herein and the nucleic acid ligands identified by such methods are useful for both therapeutic and diagnostic purposes. Therapeutic uses include the treatment or prevention of diseases or medical conditions in human patients. Many of the therapeutic uses are described in the background of the invention, particularly, nucleic acid ligands to selectins are useful as antiinflammatory agents. Antagonists to the selectins modulate extravasion of WO 96/40703 PCT/US96/09455 16 leukocytes at sites of inflammation and thereby reduce neutrophil caused host tissue damage. Diagnostic utilization may include both in vivo or in vitro diagnostic applications. The SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications. SELEX identifies nucleic acid ligands that are able to bind targets with high affinity and with surprising specificity. These characteristics are, of course, the desired properties one skilled in the art would seek in a diagnostic ligand.

The nucleic acid ligands of the present invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any nucleic acid ligand by procedures known in the art to incorporate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures. The nucleic acid ligands to lectin, particularly selectins, described herein may specifically be used for identification of the lectin proteins.

SELEX provides high affinity ligands of a target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to lectin targets. Specifically, the present invention describes the identification of nucleic acid ligands to Wheat Germ Agglutinin, and the selectins, specifically, L-selectin, P-selectin and E-selectin. In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligands to lectins are described.

In order to produce nucleic acids desirable for use as a pharmaceutical, it is preferred that the nucleic acid ligand binds to the target in a manner capable of achieving the desired effect on the target; be as small as possible to obtain the desired effect; be as stable as possible; and be a specific ligand to the chosen target. In most situations, it is preferred that the nucleic acid ligand have the highest possible affinity to the target.

In the present invention, a SELEX experiment was performed in search of nucleic acid ligands with specific high affinity for Wheat Germ Agglutinin from a degenerate library containing 50 random positions (50N). This invention includes the specific nucleic acid ligands to Wheat Germ Agglutinin shown in Table 2 (SEQ ID NOS: 4-55), identified by the methods described in Examples 1 and 2.

Specifically, RNA ligands containing 2'-NH2 modified pyrimidines are provided.

WO 96/40703 PCT/US96/09455 17 The scope of the ligands covered by this invention extends to all nucleic acid ligands of Wheat Germ Agglutinin, modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to the ligands shown in Table 2. By substantially homologous it is meant a degree of primary sequence homology in excess of most preferably in excess of 80%. A review of the sequence homologies of the ligands of Wheat Germ Agglutinin shown in Table 2 shows that sequences with little or no primary homology may have substantially the same ability to bind Wheat Germ Agglutinin. For these reasons, this invention also includes nucleic acid ligands that have substantially the same ability to bind Wheat Germ Agglutinin as the nucleic acid ligands shown in Table 2. Substantially the same ability to bind Wheat Germ Agglutinin means that the affinity is within a few orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence substantially homologous to those specifically described herein has substantially the same ability to bind Wheat Germ Agglutinin.

In the present invention, SELEX experiments were performed in search of nucleic acid ligands with specific high affinity for L-selectin from degenerate libraries containing 30 or 40 random positions (30N or 40N). This invention includes the.specific nucleic acid ligands to L-selectin shown in Tables 8, 12 and 16 (SEQ ID NOS: 67-117, 129-180, 185-196 and 293-388), identified by the methods described in Examples 7, 8, 13, 14, 22 and 23. Specifically, RNA ligands containing 2'-NH2 or 2'-F pyrimidines and ssDNA ligands are provided. The scope of the ligands covered by this invention extends to all nucleic acid ligands of L-selectin, modified and unmodified, identified according to the SELEX procedure.

More specifically, this invention includes nucleic acid sequences that are substantially homologous to the ligands shown in Tables 8, 12 and 16. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. A review of the sequence homologies of the ligands of L-selectin shown in Tables 8, 12 and 16 shows that sequences with little or no primary homology may have substantially the same ability to bind L-selectin. For these reasons, this invention also includes nucleic acid ligands that have substantially the same ability to bind L-selectin as the nucleic acid ligands shown in Tables 8, 12 and 16. Substantially the same ability to bind Lselectin means that the affinity is within a few orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in WO 96/40703 PCT/US96/09455 18 the art to determine whether a.given sequence substantially homologous to those specifically described herein has substantially the same ability to bind L-selectin.

In the present invention, SELEX experiments were performed in search of nucleic acid ligands with specific high affinity for P-selectin from degenerate libraries containing 50 random positions (50N). This invention includes the specific nucleic acid ligands to P-selectin shown in Tables 19 and 25 (SEQ ID NOS: 199- 247 and 251-290), identified by the methods described in Examples 27, 28, 35 and 36. Specifically, RNA ligands containing 2'-NH2 and 2'-F pyrimidines are provided. The scope of the ligands covered by this invention extends to all nucleic acid ligands of P-selectin, modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to the ligands shown in Tables 19 and 25. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. A review of the sequence homologies of the ligands of P-selectin shown in Tables 19 and 25 shows that sequences with little or no primary homology may have substantially the same ability to bind P-selectin. For these reasons, this invention also includes nucleic acid ligands that have substantially the same ability to bind P-selectin as the nucleic acid ligands shown in Tables 19 and 25. Substantially the same ability to bind P-selectin means that the affinity is within a few orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence substantially homologous to those specifically described herein has substantially the same ability to bind P-selectin.

In the present invention, a SELEX experiment was performed in search of nucleic acid ligands with specific high affinity for E-selectin from a degenerate library containing 40 random positions (40N). This invention includes specific nucleic acid ligands to E-selectin identified by the methods described in Example The scope of the ligands covered by this invention extends to all nucleic acid ligands of E-selectin, modified and unmodified, identified according to the SELEX procedure.

Additionally, the present invention includes multivalent Complexes comprising the nucleic acid ligands of the invention. The mulivalent Complexes increase the binding energy to facilitate better binding affinities through slower offrates of the nucleic acid ligands. The multivalent Complexes may be useful at lower doses than their monomeric counterparts. In addition, high molecular weight polyethylene glycol was included in some of the Complexes to decrease the in vivo WO 96/40703 PCT/US96/09455 19 clearance rate of the Complexes. Specifically, nucleic acid ligands to L-selectin were placed in multivalent Complexes.

As described above, because of their ability to selectively bind lectins, the nucleic acid ligands to lectins described herein are useful as pharmaceuticals. This invention, therefore, also includes a method for treating lectin-mediated diseases by administration of a nucleic acid ligand capable of binding to a lectin.

Therapeutic compositions of the nucleic acid ligands may be administered parenterally by injection, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis or suppositories, are also envisioned. One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used. In one preferred embodiment, it is envisioned that the carrier and the ligand constitute a physiologically-compatible, slow release formulation. The primary solvent in such a carrier may be either aqueous or nonaqueous in nature. In addition, the carrier may contain other pharmacologicallyacceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.

Similarly, the carrier may contain still other pharmacologically-acceptable excipients for modifying or maintaining the stability, rate of dissolution, release, or absorption of the ligand. Such excipients are those substances usually and customarily employed to formulate dosages for parental administration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready to use form or requiring reconstitution immediately prior to administration. The manner of administering formulations containing nucleic acid ligands for systemic delivery may be via subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectal suppository.

Well established animal models exist for many of the disease states which are candidates for selectin antagonist therapy. Models available for testing the efficacy of oligonucleotide selectin antagonists include: 1) mouse models for peritoneal inflammation Pizcueta and F.W.

Luscinskas, 1994, Am. J. Pathol. 145, 461-469), diabetes Hanninen et al., 1992, J. Clin. Invest. 92, 2509-2515), lymphocyte trafficking Bradley et al., 1994, J. Exp. Med., 2401-2406), glomerulonephritis Tipping et al., 1994, Kidney Int. 46, 79-88), experimental allergic encephalomyelitis Dopp et al., WO 96/40703 PCT/US96/09455 1994, J. Neuroimmunol. 54: 129-144), acute inflammation in human/SCID mouse chimera Yan et al., 1994, J. Immunol. 152, 3053-3063), endotoxinmediated inflammation Sanders et al., 1992, Blood 80, 795-800); 2) rat models for acute lung injury Milligan et al., 1994, J. Immunol.

152, 832-840), hind limb ischemia/reperfusion injury Seekamp et al., 1994, Am. J. Pathol 144, 592-598), remote lung injury Seekamp et al., 1994, supra; D.L. Carden et al., 1993, J. Appl. Physiol 75, 2529-2543), neutrophil rolling on mesenteric venules Ley et al., 1993, Blood 82, 1632-1638), myocardial infarction ischemia reperfusion injury Altavilla et al., 1994, Eur. J. Pharmacol.

Environ. Toxicol. Pharmacol. 270, 45-51); 3) rabbit models for hemorrhagic shock Winn et al., 1994, Am. J.

Physiol. Heart Circ. Physiol. 267, H2391-H2397), ear ischemia reperfusion injury Mihelcic et al., 1994, Bollod 84, 2333-2328) neutrophil rolling on mesenteric venules Olofsson et al., Blood 84, 2749-2758), experimental meningitis (C.

Granert et al., 1994, J. Clin. Invest. 93, 929-936); lung, peritoneal and subcutaneous bacterial infection Sharer et al., 1993, J. Immunol. 151, 4982- 4988), myocardial ischemia/repefusion Montrucchio et al., 1989, Am. J.

Physiol. 256, H1236-H1246), central nervous system ischemic injury Clark et al., 1991, Stroke 22, 877-883); 4) cat models for myocardial infraction ischemia reperfusion injury (M.Buerke et al., 1994, J. Pharmacol. Exp. Ther. 271, 134-142); dog models for myocardial infarction ischemia reperfusion injury(D.J.

Lefer et al., 1994, Circulation 90, 2390-2401); 6) pig models for arthritis Jamar et al., 1995, Radiology 194, 843-850); 7) rhesus monkey models for cutaneous inflammation Silber et al., Lab.

Invest. 70, 163-175); 8) cynomolgus monkey models for renal transplants Wee, 1991, Transplant. Prod. 23, 279-280); and 9) baboon models for dacron grafts Palabrica et al, 1992, Nature 359, 848-851), septic, traumatic and hypovolemic shock Redl et al., 1991, Am. J.

Pathol. 139, 461-466).

The nucleic acid ligands to lectins described herein are useful as pharmaceuticals and as diagnostic reagents.

Examples The following examples are illustrative of certain embodiments of the invention and are not to be construed as limiting the present invention in any way.

Examples 1-6 describe identification and characterization of 2'-NH2 RNA ligands to WO 96/40703 PCT/US96/09455 21 Wheat Germ Agglutinin. Examples 7-12 described identification and characterization of 2'-NH2 RNA ligands to L-selectin. Examples 13-21 describe identification and characterization of ssDNA ligands to L-selectin. Examples 22-25 describe identification and characterization of 2'-F RNA ligands to L-selectin.

Example 26 describes identification of ssDNA ligands to P-selectin. Examples 27- 39 describes identification and characterization of 2'-NH2 and 2'-F RNA ligands to P-selectin. Example 40 describes identification of nucleic acid ligands to E-selectin.

Example 1 Nucleic Acid Ligands to Wheat Germ Agglutinin The experimental procedures outlined in this Example were used to identify and characterize nucleic acid ligands to wheat germ agglutinin (WGA) as described in Examples 2-6.

Experimental Procedures A) Materials Wheat Germ Lectin (Triticum vulgare) Sepharose 6MB beads were purchased from Pharmacia Biotech. Wheat Germ Lectin, Wheat Germ Agglutinin, and WGA are used interchangeably herein. Free Wheat Germ Lectin (Triticum vulgare) and all other lectins were obtained from E Y Laboratories; methyl-ca-Dmannopyranoside was from Calbiochem and N-acetyl-D-glucosamine, GlcNAc, and the trisaccharide N N' N'-triacetylchitotriose, (GlcNAc)3, were purchased from Sigma Chemical Co. The 2'-NH2 modified CTP and UTP were prepared according to Pieken et. al. (1991, Science 253:314-317). DNA oligonucleotides were synthesized by Operon. All other reagents and chemicals were purchased from commercial sources. Unless otherwise indicated, experiments utilized Hanks' Balanced Salt Solutions (HBSS; 1.3 mM CaCl2, 5.0 mM KC1, 0.3 mM KH2PO4, mM MgC12.6H20, 0.4 mM MgSO4.7H20, 138 mM NaC1, 4.0 mM NaHCO3, 0.3 mM Na2HPO4, 5.6 mM D-Glucose; GibcoBRL).

B) SELEX The SELEX procedure is described in detail in United States Patent 5,270,163 and elsewhere. In the wheat germ agglutinin SELEX experiment, the DNA template for the initial RNA pool contained 50 random nucleotides, flanked by N9 5' and 3' fixed regions (50N9) 5' gcuccgccagagaccaaccgagaa 3' (SEQ ID NO: All C and U have 2'-NH2 substituted for 2'-OH for ribose. The primers for the PCR were the following: Primer 5' taatacgactcactatagggaaaagcgaatcatacacaaga 3' (SEQ ID NO: 2) and 3' Primer 5' ttctcggttggttctggcggagc 3' (SEQ ID NO: The fixed regions of the WO 96/40703 PCT/US96/09455 22 starting random pool include DNA primer annealing sites for PCR and cDNA synthesis as well as the consensus T7 promoter region to allow in vitro transcription. These single-stranded DNA molecules were converted into doublestranded transcribable templates by PCR amplification. PCR conditions were 50 mM KC1, 10 mM Tris-C1, pH 8.3, 0.1% Triton X-100, 7.5 mM MgC12, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of Taq DNA polymerase.

Transcription reactions contained 5 mM DNA template, 5 units/pl T7 RNA polymerase, 40 mM Tris-Cl (pH 12 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100, 4 PEG 8000, 2 mM each of 2'-OH ATP, 2'- OH GTP, 2'-NH2 CTP, 2'-NH2 UTP, and 0.31 mM a- 32 P 2'-OH ATP.

The strategy for partitioning WGA/RNA complexes from unbound RNA was 1) to incubate the RNA pool with WGA immobilized on sepharose beads; 2) to remove unbound RNA by extensive washing; and 3) to specifically elute RNA molecules bound at the carbohydrate binding site by incubating the washed beads in buffer containing high concentrations of (GlcNAc)3. The SELEX protocol is outlined in Table 1.

The WGA density on Wheat Germ Lectin Sepharose 6MB beads is approximately 5 mg/ml of gel or 116 pM (manufacturer's specifications). After extensive washing in HBSS, the immobilized WGA was incubated with RNA at room temperature for 1 to 2 hours in a 2 ml siliconized column with constant rolling (Table Unbound RNA was removed by extensive washing with HBSS. Bound RNA was eluted as two fractions; first, nonspecifically eluted RNA was removed by incubating and washing with 10 mM methyl-a-D-mannopyranoside in HBSS (Table 1; rounds 1-4) or with HBSS (Table 1; rounds 5-11); second, specifically eluted RNA was removed by incubating and washing with 0.5 to 10 mM (GlcNAc)3 in HBSS (Table The percentage of input RNA that was specifically eluted is recorded in Table 1.

The specifically eluted fraction was processed for use in the following round. Fractions eluted from immobilized WGA were heated at 90 C for 5 minutes in 1% SDS, 2% P-mercaptoethanol and extracted with phenol/chloroform. RNA was reverse transcribed into cDNA by AMV reverse transcriptase at 48 C for min in 50 mM Tris-Cl pH 60 mM NaC1, 6 mM Mg(OAc)2, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/tl AMV RT. PCR amplification of this cDNA resulted in approximately 500 pmol double-stranded DNA, transcripts of which were used to initiate the next round of SELEX.

WO 96/40703 PCTIUS96/09455 23 D) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to determine the affinity of RNA ligands for WGA and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 pm pore size, Millipore; or pure nitrocellulose, 0.45 prn pore size, Bio-Rad) were placed on a vacuum manifold and washed with 4 ml of HBSS buffer under vacuum.

Reaction mixtures, containing 3 2 P labeled RNA pools and unlabeled WGA, were incubated in HBSS for 10 min at room temperature, filtered, and then immediately washed with 4 ml HBSS. The filters were air-dried and counted in a Beckman LS6500 liquid scintillation counter without fluor.

WGA is a homodimer, molecular weight 43.2 kD, with 4 GlcNAc binding sites per dimer. For affinity calculations, we assume one RNA ligand binding site per monomer (two per dimer). The monomer concentration is defined as 2 times the dimer concentration. The equilibrium dissociation constant, Kd, for an RNA pool or specific ligand that binds monophasically is given by the equation Kd [Pf[Rf]/[RP] where, [RfJ free RNA concentration [Pf] free WGA monomer concentration concentration of RNA/WGA monomer complexes Kd dissociation constant A rearrangement of this equation, in which the fraction of RNA bound at equilibrium is expressed as a function of the total concentration of the reactants, was used to calculate Kds of monophasic binding curves: q (PT RT Kd ((PT RT Kd) 2 -4 PT RT) 1 2 q fraction of RNA bound [PT] total WGA monomer concentration [RT] total RNA concentration Kds were determined by least square fitting of the data points using the graphics program Kaleidagraph (Synergy Software, Reading PA).

E) Cloning and Sequencing The sixth and eleventh round PCR products were re-amplified with primers which contain a BamH1 or a EcoRI restriction endonuclease recognition site. Using these restriction sites the DNA sequences were inserted directionally into the pUC18 vector. These recombinant plasmids were transformed into E. coli strain JM109 (Stratagene, La Jolla, CA). Plasmid DNA was prepared according to the alkaline WO 96/40703 PCT/US96/09455 24 hydrolysis method (Zhou et al., 1990 Biotechniques 8:172-173) and about 72 clones were sequenced using the Sequenase protocol (United States Biochemical Corporation, Cleveland, OH). The sequences are provided in Table 2.

F) Competitive Binding Studies Competitive binding experiments were performed to determine if RNA ligands and (GlcNAc)3 bind the same site on WGA. A set of reaction mixtures containing a 3 2 P labeled RNA ligand and unlabeled WGA, each at a fixed concentration (Table was incubated in HBSS for 15 min at room temperature with (GlcNAc)3. Individual reaction mixtures were then incubated with a (GlcNAc)3 dilution from a 2-fold dilution series for 15 minutes. The final (GlcNAc)3 concentrations ranged from 7.8 pM to 8.0 mM (Table The reaction mixtures were filtered, processed and counted as described in "Nitrocellulose Filter Binding Assay," paragraph D above.

Competition titration experiments were analyzed by the following equation to determine the concentration of free protein as a function of the total concentration of competitor added, [CT]: 0

+KC[P]))-PT

where LT is 'the concentration of initial ligand, KL is the binding constant of species L to the protein (assuming 1:1 stiochiometry) and Kc is the binding constant of species C to the protein (assuming 1:1 stiochiometry). Since it is difficult to obtain a direct solution for equation 1, iteration to determine values of to a precision of lxlO- 1 5 was used. Using these values of the concentration of protein-ligand complex [PL] as a function of [CT] was determined by the following equation: [PL] KL[LT][P](1+KL[P]) Since the experimental data is expressed in terms of the calculated concentration of [PL] was normalized by the initial concentration of [PLo] before addition of the competitor. ([PLo] was calculated using the quadratic solution for the standard binding equation for the conditions used. The maximum and minimum was allowed to float during the analysis as shown in the following equation.

WO 96/40703 PCT/US96/09455 A non-linear least-squares fitting procedure was used as described by P.R.

Bevington (1969) Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill publishers. The program used was originally written by Stanley J.

Gill in MatLab and modified for competition analysis by Stanley C. Gill. The data were fit to equations 1-3 to obtain best fit parameters for KC, M and B as a function of [CT] while leaving KL and PT fixed.

G) Inhibition of WGA Agglutinating Activity Agglutination is a readily observed consequence of the interaction of a lectin with cells and requires that individual lectin molecules crosslink two or more cells.

Lectin mediated agglutination can be inhibited by sugars with appropriate specificity.

Visual assay of the hemagglutinating activity of WGA and the inhibitory activity of RNA ligands, GlcNAc and (GlcNAc)3 was made in Falcon round bottom 96 well microtiter plates, using sheep erythrocytes. Each well contained 54 il of erythrocytes (2.5 x 108 cells/ml) and 54 pl of test solution.

To titrate WGA agglutinating activity, each test solution contained a WGA dilution from a 4-fold dilution series. The final WGA concentrations ranged from 0.1 pM to 0.5 pM. For inhibition assays, the test solutions contained 80 nM WGA (monomer) and a dilution from a 4-fold dilution series of the designated inhibitor.

Reaction mixtures were incubated at room temperature for 2 hours, after which time no changes were observed in the precipitation patterns of erythrocytes. These experiments were carried out in Gelatin Veronal Buffer (0.15 mM CaCl2, 141 mM NaCI, 0.5 mM MgC12, 0.1% gelatin, 1.8 mM sodium barbital, and 3.1 mM barbituric acid, pH 7.3 Sigma #G-6514).

In the absence of agglutination, erythrocytes settle as a compact pellet.

Agglutinated cells form a more diffuse pellet. Consequently, in visual tests, the diameter of the pellet is diagnostic for agglutination. The inhibition experiments included positive and negative controls for agglutination and appropriate controls to show that the inhibitors alone did not alter the normal precipitation pattern.

Example 2 RNA Ligands to WGA A. SELEX The starting RNA library for SELEX, randomized 50N9 (SEQ ID NO: 1), contained approximately 2 x 1015 molecules (2 nmol RNA). The SELEX protocol is outlined in Table 1. Binding of randomized RNA to WGA is undetectable at 36 pM WGA monomer. The dissociation constant of this interaction is estimated to be >4 mM.

WO 96/40703 PCT/US96/09455 26 The percentage of input RNA eluted by (GlcNAc)3 increased from 0.05 in the first round, to 28.5 in round 5 (Table The bulk Kd of round 5 RNA was 600 nM (Table Since an additional increase in specifically eluted RNA was not observed in round 6a (Table round 6 was repeated (Table 1, round 6b) with two modifications to increase the stringency of selection: the volume of gel, and hence the mass of WGA, was reduced ten fold; and RNA was specifically eluted with increasing concentrations of (GlcNAc)3, in stepwise fashion, with only the last eluted RNA processed for the following round. The percentage of specifically eluted RNA increased from 5.7 in round 6b to 21.4 in round 8, with continued improvement in the bulk Kd (260 nM, round 8 RNA, Table 1).

For rounds 9 through 11, the WGA mass was again reduced ten fold to further increase stringency. The Kd of round 11 RNA was 68 nM. Sequencing of the bulk starting RNA pool and sixth and eleventh round RNA revealed some nonrandomness in the variable region at the sixth round and increased nonrandomess at round eleven.

To monitor the progess of SELEX, ligands were cloned and sequenced from round 6b and round 11. From each of the two rounds, 36 randomly picked clones were sequenced. Sequences were aligned manually and are shown in Table 2.

B. RNA Sequences From the sixth and eleventh rounds, respectively, 27 of 29 and 21 of sequenced ligands were unique. The number before the in the ligand name indicates whether it was cloned from the round 6 or round 11 pool. Only a portion of the entire clone is shown in Table 2 (SEQ ID NOS: 4-55). The entire evolved random region is shown in upper case letters. Any portion of the fixed region is shown in lower case letters. By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides. In Table 2, ligands sequences are shown in standard single letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). Sequences that were isolated more than once are indicated by the parenthetical number, following the ligand isolate number. These clones fall into nine sequence families (1 9) and a group of unrelated sequences (Orphans).

The distribution of families from round six to eleven provides a clear illustration of the appearance and disappearance of ligand families in response to increased selective pressure (Table Family 3, predominant (11/29 ligands) in round 6, has nearly disappeared (2/35) by round 11. Similarly, minor families 6 WO 96/40703 PCT/US96/09455 27 through 9 virtually disappear. In contrast, only one (family 1) of round eleven's predominant families 2, 4 and 5) was detected in round six. The appearance and disappearance of families roughly correlates with their binding affinities.

Alignment (Table 2) defines consensus sequences for families 1-4 and 6-9 (SEQ ID NOS: 56-63). The consensus sequences of families 1-3 are long (20, 16 and 16, respectively) and very highly conserved. The consensus sequences of families 1 and 2 contain two sequences in common: the trinucleotide TCG and the pentanucleotide ACGAA. A related tetranucleotide, AACG, occurs in family 3. The variation in position of the consensus sequences within the variable regions indicates that the ligands do not require a specific sequence from either the 5' or 3' fixed region.

The consensus sequences of family 1 and 2 are flanked by complementary sequences 5 or more nucleotides in length. These complementary sequences are not conserved and the majority include minor discontinuities. Family 3 also exhibits flanking complementary sequences, but these are more variable in length and structure and utilize two nucleotide pairs of conserved sequence.

Confidence in the family 4 consensus sequence (Table 2) is limited by the small number of ligands, the variability of spacing and the high G content. The pentanucleotide, RCTGG, also occurs in families 5 and 8. Ligands of family 5 show other sequence similarities to those of family 4, especially to ligand 11.28.

C. Affinities The dissociation constants for representative members of families 1-9 and orphan ligands were determined by nitrocellulose filter binding experiments and are listed in Table 3. These calculations assume one RNA ligand binding site per WGA monomer. At the highest WGA concentration tested (36 pM WGA monomer), binding of random RNA is not observed, indicating a Kd at least 100-fold higher than the protein concentration or 4 mM.

The data in Table 3 define several characteristics of ligand binding. First, RNA ligands to WGA bind monophasically. Second, the range of measured dissociation constants is 1.4 nM to 840 nM. Third, the binding for a number of ligands, most of which were sixth round isolates, was less than 5% at the highest WGA concentration tested. The dissociation constants of these ligands are estimated to be greater than 20 pM. Fourth, on average eleventh round isolates have higher affinity than those from the sixth round. Fifth, the SELEX probably was not taken to completion; the best ligand (11.20)(SEQ ID NO: 40) is not the dominant species.

Since the SELEX was arbitrarily stopped at the 11th round, it is not clear that 11.20 WO 96/40703 PCT/US96/09455 28 would be the ultimate winner. Sixth, even though the SELEX was not taken to completion, as expected, RNA ligands were isolated that bind WGA with much greater affinity than do mono- or oligosaccharides the affinity of 11.20 is 5x10 greater than that of GlcNAc, Kd 760 pM, and 850 better than that of (GlcNAc)3, Kd 12 pM; Y.Nagata and M.Burger, 1974, supra). This observation validates the proposition that competitive elution allows the isolation of oligonucleotide ligands with affinities that are several orders of magnitude greater than that of the competing sugar.

In addition these data show that even under conditions of high target density, 116 pmol WGA dimer/lp of beads, it is possible to overcome avidity problems and recover ligands with nanomolar affinities. From the sixth to the eleventh round (Table in response to increased selective pressure as indicated by the improvement in bulk Kd (Table sequence families with lower than average affinity (Table 3) are eliminated from the pool.

Example 3 Specificity of RNA Ligands to WGA The affinity of WGA ligands 6.8, 11.20 and 11.24 (SEQ ID NOS: 13, and 19) for GlcNAc binding lectins from Ulex europaeus, Datura stramonium and Canavalia ensiformis were determined by nitrocellulose partitioning. The results of this determination are shown in Table 4. The ligands are highly specific for WGA.

For example, the affinity of ligand 11.20 for WGA is 1,500, 8,000 and >15,000 fold greater than it is for the U. europaeus, D. stramonium and C. ensiformis lectins, respectively. The 8,000 fold difference in affinity for ligand 11.20 exhibited by T. vulgare and D. stramonium compares to a 3 to 10 fold difference in their affinity for oligomers of GlcNAc and validates the proposition that competitive elution allows selection of oligonucleotide ligands with much greater specificity than monomeric and oligomeric saccharides (J.F.Crowley et al., 1984, Arch. Biochem.

and Biophys. 231:524-533; Y.Nagata and M.Burger, 1974, supra; J-P.Privat et al., FEBS Letters 46:229-232).

Example 4 Competitive Binding Studies If an RNA ligand and a carbohydrate bind a common site, then binding of the RNA ligand is expected to be competitively inhibited by the carbohydrate.

Furthermore, if the oligonucleotide ligands bind exclusively to carbohydrate binding sites, inhibition is expected to be complete at high carbohydrate concentrations. In the experiments reported in Table 5, dilutions of unlabeled (GlcNAc)3, from a 2- WO 96/40703 PCT/US96/09455 29 fold dilution series, were added to three sets of binding reactions that contained WGA and an a- 32 P labeled RNA ligand 11.20 or 11.24 (SEQ ID NOS: 13, andl9); [RNA] final [WGA]final 15 nM). After a 15 minute incubation at room temperature, the reactions were filtered and processed as in standard binding experiments.

Qualitatively, it is clear that RNA ligands bind only to sites at which (GlcNAc)3 binds, since inhibition is complete at high (GlcNAc)3 concentrations (Table These data do not rule out the possibility that (GlcNAc)3 binds one or more sites that are not bound by these RNA ligands.

Quantitatively, these data fit a simple model of competitive inhibition (Table 5) and give estimates of 8.4, 10.9 and 19.4 pM for the Kd of (GlcNAc)3. These estimates are in good agreement with literature values (12 pM 4 C, Nagata and Burger, 1974, supra; 11 pM 10.8 C, Van Landschoot et al., 1977, Eur. J.

Biochem. 79:275-283; 50 M, M.Monsigny et al., 1979, Eur J. Biochem. 98:39- These data confirm the proposition that competitive elution with a specific carbohydrate targets the lectin's carbohydrate binding site.

Example Inhibition of WGA Agglutinating Activity At 0.5 pM, RNA ligands 6.8 and 11.20 (SEQ ID NO: 13 and 40) completely inhibit WGA mediated agglutination of sheep erythrocytes (Table Ligand 11.24 (SEQ ID NO: 19) is not as effective, showing only partial inhibition at 2 pM, the highest concentration tested (Table (GlcNAc)3 and GlcNAc completely inhibit agglutination at higher concentrations, 8 M and 800 pM, respectively, (Table 6; Monsigny et al., supra). The inhibition of agglutination varifies the proposition that ligands isolated by this procedure will be antagonists of lectin function. Inhibition also suggests that more than one RNA ligand is bound per WGA dimer, since agglutination is a function of multiple carbohydrate binding sites.

An alternative interpretation for the inhibition of agglutination is that charge repulsion prevents negatively charged WGA/RNA complexes from binding carbohydrates (a necessary condition for agglutination) on negatively charged cell surfaces. This explanation seems unlikely for two reasons. First, negatively charged oligonucleotide ligands selected against an immobilized purified protein are known to bind to the protein when it is presented in the context of a cell surface (see Example 10, L-selectin cell binding). Second, negatively charged (p 4) succinylated WGA is as effective as native WGA (pI 8.5) in agglutinating erythrocytes (M.Monsigny et al., supra).

WO 96/40703 PCT/US96/09455 Example 6 Secondary Structure of High Affinity WGA Ligands In favorable instances, comparative analysis of aligned sequences allows deduction of secondary structure and structure-function relationships. If the nucleotides at two positions in a sequence covary according to Watson-Crick base pairing rules, then the nucleotides at these positions are apt to be paired.

Nonconserved sequences, especially those that vary in length are not apt to be directly involved in function, while highly conserved sequence are likely to be directly involved.

Comparative analyses of both family 1 and 2 sequences each yield a hairpin structure with a large, highly conserved loop (Figures la and lb). Interactions between loop nucleotides are likely but they are not defined by these data. The stems of individual ligands vary in sequence, length and structure a variety of bulges and internal loops are allowed; Table Qualitatively it is clear that the stems are validated by Watson/Crick covariation and that by the rules of comparative analysis the stems are not directly involved in binding WGA. Family 3 can form a similar hairpin in which 2 pairs of conserved nucleotides are utilized in the stem (Figure lc).

If it is not possible to fold the ligands of a sequence family into homologous structures, their assignment to a single family is questionable. Both ligand 11.7, the dominant member of family 4, and ligand 11.28 can be folded into two plane G-quartets. However, this assignment is speculative: 1) 11.28 contains five GG dinucleotides and one GGGG tetranucleotide allowing other G-quartets; and 2) ligands 11.2 and 11.33 cannot form G-quartets. On the other hand, all ligands can form a hairpin with the conserved sequence GAGRFTNCRT in the loop. However, the conserved sequence RCTGGC (Table 2) does not have a consistent role in these hairpins.

Multiple G-quartet structures are possible for Family 5. One of these resembles the ligand 11.7 G-quartet. No convincing hairpin structures are possible for ligand 11.20.

Example 7 2'-NH? RNA Ligands to Human L-Selectin The experimental procedures outlined in this Example were used to identify and characterize the 2'-NH2 RNA ligands to human L-selectin in Examples 8-12.

Experimental Procedures WO 96/40703 PCT/US96/09455 31 A) Materials LS-Rg is a chimeric protein in which the extracellular domain of human Lselectin is joined to the Fc domain of a human G2 immunoglobulin (Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg, PS-Rg and CD22P-Rg are analogous constructs of E-selectin, P-selectin and CD223 joined to a human G1 immunoglobulin Fc domain Nelson et al., 1993, supra; I. Stamenkovic et al., 1991, Cell 66, 1133-1144). Purified chimera were provided by A.Varki. Soluble P-selectin was purchased from R&D Systems. Protein A Sepharose 4 Fast Flow beads were purchased from Pharmacia Biotech. Anti-L-selectin monoclonal antibodies: SK 1 was obtained from Becton-Dickinson, San Jose, CA; DREG-56, an L-selectin specific monoclonal antibody, was purchased from Endogen, Cambridge, MA. The 2'-NH2 modified CTP and UTP were prepared according to Pieken et. al. (1991, Science 253:314-317). DNA oligonucleotides were synthesized by Operon. All other reagents and chemicals were purchased from commercial sources. Unless otherwise indicated, experiments utilized HSMC buffer (1 mM CaCl2, 1 mM MgC12, 150 mM NaCI, 20.0 mM HEPES, pH 7.4).

B) SELEX The SELEX procedure is described in detail in United States Patent 5,270,163 and elsewhere. The nucleotide sequence of the synthetic DNA template for the LS-Rg SELEX was randomized at 40 positions. This variable region was flanked by N7 5' and 3' fixed regions (40N7). 40N7 transcript has the sequence gggaggacgaugcgg-40N-cagacgacucgcccga 3' (SEQ ID NO: 64). All C and U have 2'-NH2 substituted for 2'-OH on the ribose. The primers for the PCR were the following: N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3' (SEQ ID NO: N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66) The fixed regions include primer annealing sites for PCR and cDNA synthesis as well as a consensus T7 promoter to allow in vitro transcription. The initial RNA pool was made by first Klenow extending 1 nmol of synthetic single stranded DNA and then transcribing the resulting double stranded molecules with T7 RNA polymerase. Klenow extension conditions: 3.5 nmols primer 5N7, 1.4 nmols 40N7, 1X Klenow Buffer, 0.4 mM each of dATP, dCTP, dGTP and dTTP in a reaction volume of 1 ml.

WO 96/40703 PCT/US96/09455 32 For subsequent rounds, eluted RNA was the template for AMV reverse transcriptase mediated synthesis of single-stranded cDNA. These single-stranded DNA molecules were converted into double-stranded transcription templates by PCR amplification. PCR conditions were 50 mM KC1, 10 mM Tris-C1, pH 8.3, 7.5 mM MgC12, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of Taq DNA polymerase. Transcription reactions contained 0.5 mM DNA template, 200 nM T7 RNA polymerase, 80 mM HEPES (pH 12 mM MgC12, 5 mM DTT, 2 mM spermidine, 2 mM each of 2'-OH ATP, 2'-OH GTP, 2'-NH2 CTP, 2'-NH2 UTP, and 250 nM a- 3 2 P 2'-OH ATP.

The strategy for partitioning LS-Rg/RNA complexes from unbound RNA is outlined in Tables 7a and 7b. First, the RNA pool was incubated with LS-Rg immobilized on protein A sepharose beads in HSMC buffer. Second, the unbound RNA was removed by extensive washing. Third, the RNA molecules bound at the carbohydrate binding site were specifically eluted by incubating the washed beads in HMSC buffer containing 5 mM EDTA in place of divalent cations. The 5 mM elution was followed by a non-specific 50 mM EDTA elution. LS-Rg was coupled to protein A sepharose beads according to the manufacturer's instructions (Pharmacia Biotech).

The 5 mM EDTA elution is a variation of a specific site elution strategy.

Although it is not a priori as specific as elution by carbohydrate competition, it is a general strategy for C-type (calcium dependent binding) lectins and is a practical alternative when the cost and/or concentration of the required carbohydrate competitor is unreasonable (as is the case with sialyl-Lewisx). This scheme is expected to be fairly specific for ligands that form bonds with the lectin's bound Ca because the low EDTA concentration does not appreciably increase the buffer's ionic strength and the conformation of C-type lectins is only subtly altered in the absence of bound calcium (unpublished observations cited by K. Drickamer, 1993, Biochem. Soc. Trans. 21:456-459).

In the initial SELEX rounds, which were performed at 4 OC, the density of immobilized LS-Rg was 16.7 pmols/tl of Protein A Sepharose 4 Fast Flow beads.

In later rounds, the density of LS-Rg was reduced (Tables 7a and 7b), as needed, to increase the stringency of selection. At the seventh round, the SELEX was branched and continued in parallel at 4 °C (Table 7a) and at room temperature (Table 7b). Wash and elution buffers were equilibrated to the relevant incubation temperature. Beginning with the fifth round, SELEX was often done at more than WO 96/40703 PCT1US96/09455 33 one LS-Rg density. In each branch, the eluted material from only one LS-Rg density was carried forward.

Before each round, RNA was batch adsorbed to 100 pl of protein A sepharose beads for 1 hour in a 2 ml siliconized column. Unbound RNA and RNA eluted with minimal washing (two volumes) were combined and used for SELEX input material. For SELEX, extensively washed, immobilized LS-Rg was batch incubated with pre-adsorbed RNA for 1 to 2 hours in a 2 ml siliconized column with constant rocking. Unbound RNA was removed by extensive batch washing (200 to 500 til HSMC/wash). Bound RNA was eluted as two fractions; first, bound RNA was eluted by incubating and washing columns with 5 mM EDTA in HSMC without divalent cations; second, the remaining elutable RNA was removed by incubating and/or washing with 50 mM EDTA in HSMC without divalents. The percentage of input RNA that was eluted is recorded in Tables 7a and 7b. In every round, an equal volume of protein A sepharose beads without LS-Rg was treated identically to the SELEX beads to determine background binding. All unadsorbed, wash and eluted fractions were counted in a Beckman LS6500 scintillation counter in order to monitor each round of SELEX.

The eluted fractions were processed for use in the following round (Tables 7a and 7b). After extracting with phenol/chloroform and precipitating with isopropanolethanol the RNA was reverse transcribed into cDNA by AMV reverse transcriptase either 1) at 48 C for 15 minutes and then 65 *C for minutes or 2) at 37 °C and 48 OC for 15 minutes each, in 50 mM Tris-C1 pH mM NaC1, 6 mM Mg(OAc)2, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/pl AMV RT. Transcripts of the PCR product were used to initiate the next round of SELEX.

C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to determine the affinity of RNA ligands for LS-Rg and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 tm pore size, Millipore) were placed on a vacuum manifold and washed with 2 ml of HSMC buffer under vacuum. Reaction mixtures, containing 32 P labeled RNA pools and unlabeled LS-Rg, were incubated in HSMC for 10 20 min at 4 oC, room temperature or 37 filtered, and then immediately washed with 4 ml HSMC at the same temperature. The filters were air-dried and counted in a Beckman LS6500 liquid scintillation counter without fluor.

WO 96/40703 PCT/US96/09455 34 LS-Rg is a dimeric protein that is the expression product of a recombinant gene constructed by fusing the DNA sequence that encodes the extracellular domains of human L-selectin to the DNA that encodes a human IgG2 Fc region. For affinity calculations, we assume one RNA ligand binding site per LS-Rg monomer (two per dimer). The monomer concentration is defined as 2 times the LS-Rg dimer concentration. The equilibrium dissociation constant, Kd, for an RNA pool or specific ligand that binds monophasically is given by the equation Kd [Pf][Rf]/[RP] where, [Rf] free RNA concentration [Pf] free LS-Rg monomer concentration concentration of RNA/LS-Rg complexes Kd dissociation constant A rearrangement of this equation, in which the fraction of RNA bound at equilibrium is expressed as a function of the total concentration of the reactants, was used to calculate Kds of monophasic binding curves: q (PT RT Kd ((PT RT Kd) 2 4 PT RT) 1 2 q fraction of RNA bound [PT] 2 x (total LS-Rg concentration) [RT] total RNA concentration Many ligands and evolved RNA pools yield biphasic binding curves. Biphasic binding can be described as the binding of two affinity species that are not in equilibrium. Biphasic binding data were evaluated with the equation q 2Pt+Rt+Kdl+Kd2-[(Pt+X 1R1+Kdl) 2 -4PtX1Rt]1/2 -[(Pt+X2Rt+Kd2) 2 -4PtX2Rt] 1/2, where Xi and X2 are the mole fractions of affinity species R1 and R2 and Kdl and Kd2 are the corresponding dissociation constants. Kds were determined by least square fitting Kds were determined by least square fitting of the data points using the graphics program Kaleidagraph (Synergy Software, Reading, PA).

D) Cloning and Sequencing Sixth, thirteenth (RT) and fourteenth (4 round PCR products were reamplified with primers which contain either a BamHI or a HinDIII restriction endonuclease recognition site. Using these restriction sites, the DNA sequences were inserted directionally into the pUC9 vector. These recombinant plasmids were transformed into E. coli strain DH5a (Life Technologies, Gaithersburg, MD).

WO 96/40703 PCT/US96/09455 Plasmid DNA was prepared according to the alkaline hydrolysis method (PERFECTprep, Boulder, CO). Approximately 150 clones were sequenced using the Sequenase protocol (Amersham, Arlington Heights, IL). The resulting ligand sequences are shown in Table 8.

E) Cell Binding Studies The ability of evolved ligand pools and cloned ligands to bind to L-selectin presented in the context of a cell surface was tested in experiments with isolated human peripheral blood mononuclear cells (PBMCs). Whole blood, collected from normal volunteers, was anticoagulated with 5 mM EDTA. Six milliliters of blood were layered on a 6 ml Histopaque gradient in 15 ml polypropylene tube and centrifuged (700 g) at room temperature for 30 minutes. The mononuclear cell layer was collected, diluted in 10 ml of Ca++/Mg++-free DPBS Gibco 14190- 029) and centrifuged (225 g) for 10 minutes at room temperature. Cell pellets from two gradients were combined, resuspended in 10 ml of DPBS(-) and recentrifuged as described above. These pellets were resuspended in 100 .l of SMHCK buffer supplemented with 1% BSA. Cells were counted in a hemocytometer, diluted to 2x10 7 cells/ml in SMHCK/1% BSA and immediately added to binding assays. Cell viability was monitored by trypan blue exclusion.

For cell binding assays, a constant number of cells were titrated with increasing concentrations of radiolabeled ligand. The test ligands were serially diluted in DPBS(-)/1%BSA to 2-times the desired final concentration approximately minutes before use. Equal volumes (25 p1) of each ligand dilution and the cell suspension (2x10 7 cells/ml) were added to 0.65 ml eppendorf tubes, gently vortexed and incubated on ice for 30 minutes. At 15 minutes the tubes were revortexed. The ligand/PBMC suspension was layered over 50 tl of ice cold phthalate oil (1:1 dinonyl:dibutyl phthalate) and microfuged (14,000 g) for minutes at 4 Tubes were frozen in dry ice/ethanol, visible pellets amputated into scintillation vials and counted in Beckman LS6500 scintilation counter as described in Example 7, paragraph C.

The specificity of binding to PBMCs was tested by competition with the Lselectin specific blocking monoclonal antibody, DREG-56, while saturability of binding was tested by competition with unlabeled RNA. Experimental procedure and conditions were like those for PBMC binding experiments, except that the radiolabeled RNA ligand (final concentration 5 nM) was added to serial dilutions of the competitor before mixing with PBMCs.

WO 96/40703 PCT/US96/09455 36 F) Inhibition of Selectin Binding to sialyl-Lewisx The ability of evolved RNA pools or cloned ligands to inhibit the binding of LS-Rg to sialyl-Lewisx was tested in competive ELISA assays Foxall et al., 1992, supra). For these assays, the wells of Coming (25801) 96 well microtiter plates were coated with 100 ng of a sialyl-LewisX/BSA conjugate, air dried overnight, washed with 300 gl of PBS(-) and then blocked with 1% BSA in SHMCK for 60 min at room temperature. RNA ligands were incubated with LS-Rg in SHMCK/1% BSA at room temperature for 15 min. After removal of the blocking solution, 50 pl of LS-Rg (10nM) or a LS-Rg (10nM)/RNA ligand mix was added to the coated, blocked wells and incubated at room temperature for 60 minutes. The binding solution was removed, wells were washed with 300 pl of PBS(-) and then probed with HRP conjugated anti-human IgG, at room temperature to quantitate LS- Rg binding. After a 30 minute incubation at room temperature in the dark with OPD peroxidase substrate (Sigma P9187), the extent of LS-Rg binding and percent inhibition was determined from the OD450.

Example 8 2'-NH 2 RNA Ligands to Human L-selectin A. SELEX The'starting RNA pool for SELEX, randomized 40N7 (SEQ ID NO: 63), contained approximately 1015 molecules (1 nmol RNA). The SELEX protocol is outlined in Tables 7a and 7b and Example 7. The dissociation constant of randomized RNA to LS-Rg is estimated to be approximately 10 gM. No difference was observed in the RNA elution profiles with 5 mM EDTA from SELEX and background beads for rounds 1 and 2, while the 50 mM elution produced a 2-3 fold excess over background (Table 7a). The 50 mM eluted RNA from rounds 1 and 2 were amplified for the input material for rounds 2 and 3, respectively. Beginning in round 3, the 5 mM elution from SELEX beads was significantly higher than background and was processed for the next round's input RNA. The percentage of input RNA eluted by 5 mM EDTA increased from 0.5 in the first round to 8.4 in round 5 (Table 7a). An additional increase in specifically eluted RNA from the pM LS-Rg beads was not observed in round 6 (Table 7a). To increase the stringency of selection, the density of immobilized LS-Rg was reduced ten fold in round 5 with further reductions in protein density at later rounds. The affinity of the selected pools rapidly increased and the pools gradually evolved biphasic binding characteristics.

WO 96/40703 PCT/US96/09455 37 Binding experiments with 6th round RNA revealed that the affinity of the evolving pool for L-selectin was temperature sensitive. Beginning with round 7, the SELEX was branched; one branch was continued at 4 °C (Table 7a) while the other was conducted at room temperature (Table 7b). Bulk sequencing of 6th, 13th (rm temp) and 14th (4 RNA pools revealed noticeable non-randomness at round six and dramatic non-randomess at the later rounds. The 6th round RNA bound monophasically at 4 °C with a dissociation constant of approximately 40 nM, while the 13th and 14th round RNAs bound biphasically with high affinity Kds of approximately 700 pM. The molar fraction of the two pools that bound with high affinity were 24 and 65 respectively. The binding of all tested pools required divalent cations. In the absence of divalent cations, the Kds of the 13th and 14th round pools increased to 45 nM and 480 nM, respectively (HSMC, minus Ca plus 2 mM EDTA).

To monitor the progress of SELEX, ligands were cloned and sequenced from rounds 6, 13 (rm temp) and 14 (4 Sequences were aligned manually and with the aid of a computer program that determines consensus sequences from frequently occurring local alignments.

B. Sequences In Table 8, ligand sequences are shown in standard single letter code (Comish-Bowden, 1985 NAR 13: 3021-3030). The letter/number combination before the in the ligand name indicates whether it was cloned from the round 6, 13 or 14 pools. Only the evolved random region is shown in Table 8. Any portion of the fixed region is shown in lower case letters. By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise. From the sixth, thirteenth and fourteenth rounds, respectively, 26 of 48, 8 of 24 and 9 of 70 sequenced ligands were unique. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides.

Sequences that were isolated more than once, are indicated by the parenthetical number, following the ligand isolate number. These clones fall into thirteen sequence families (I XIII) and a group of unrelated sequences (Orphans)(SEQ ID NOs: 67-117).

Two families, I and IIl, are defined by ligands from multiple lineages. Both families occur frequently in round 6, but only one family III ligand was identified in the final rounds. Six families (IV, V, VI, VII, VIII, and possibly II) are each defined by just two lineages which limits confidence in their consensus sequences.

WO 96/40703 PCT/US96/09455 38 Five families (IX through XII) are defined by a single lineage which precludes determination of consensus sequences.

Ligands from family II dominate the final rounds: 60/70 ligands in round 14 and 9/24 in round 13. Family II is represented by three mutational variations of a single sequence. One explanation for the recovery of a single lineage is that the ligand's information content is extremely high and was therefore represented by a unique species in the starting pool. Family II ligands were not detected in the sixth round which is consistent with a low frequency in the initial population. An alternative explanation is sampling error. Note that a sequence of questionable relationship was detected in the sixth round.

The best defined consensus sequences are those of family I, AUGUGUA (SEQ ID NO: 118), and of family m, AACAUGAAGUA (SEQ ID NO: 120), as shown in Table 8. Family II has two additional, variably spaced sequences, AGUC and ARUUAG, that may be conserved. The tetranucleotide AUGW is found in the consensus sequence of families I, m, and VII and in families II, VI and IX. If this sequence is significant, it suggests that the conserved sequences of ligands of family VIII are circularly permuted. The sequence AGAA is found in the consensus sequence of families IV and VI and in families X and XIII.

C. Affinities The dissociation constants for representative ligands from rounds 13 and 14, including all orphans, were determined by nitrocellulose filter binding experiments are described in Example 7 and the results are listed in Table 9. These calculations assume two RNA ligand binding sites per chimera. The affinity of random RNA cannot be reliably determined but is estimated to be approximately 10 pM.

In general, ligands bind monophasically with dissociation constants ranging from 50 pM to 15 nM at 4 Some of the highest affinity ligands bind biphasically. Although ligands of families I, VII, X and orphan F14.70 bind about equally well at 4 OC and room temperature, in general the affinities decrease with increasing temperature. The observed affinities substantiate the proposition that it is possible to isolate oligonucleotide ligands with affinities that are several orders of magnitude greater than that of carbohydrate ligands.

WO 96/40703 PCT/US96/09455 39 Example 9 Specificity of 2'-NH9 RNA Ligands to L-Selectin The affinity of L-selectin ligands to ES-Rg, PS-Rg and CD22p-Rg were determined by nitrocellulose partitioning as described in Example 7. As indicated in Table 10, the ligands are highly specific for L-selectin. In general, a ligand's affinity for ES-Rg is 10 3 -fold lower and that for PS-Rg is about 10 4 -fold less than for LS-Rg. Binding above background is not observed for CD22p-Rg at the highest protein concentration tested (660 nM), indicating that ligands do not bind the Fc domain of the chimeric constructs nor do they have affinity for the sialic acid binding site of an unrelated lectin. The specificity of oligonucleotide ligand binding contrasts sharply with the binding of cognate carbohydrates by the selectins and confirms the proposition that SELEX ligands will have greater specificity than carbohydrate ligands.

Example Binding of L-Selectin 2'-NH? RNA Ligands to Human PBMCs Since the L-selectin ligands were isolated against purified, immobilized protein, it is essential to demonstrate that they bind L-selectin presented in the context of a cell surface. Comparison of 2nd and 9th round RNAs (Figure 2) shows that the evolved (9th round) ligand pool binds isolated PBMCs with high affinity and, as expected for specific binding, in a saturable fashion. The binding of round 2 RNA appears to be non-saturable as is characteristic of non-specific binding. The cloned ligand, F14.12 (SEQ ID NO: 78), also binds in a saturable fashion with a dissociation constant of 1.3 nM, while random 40N7 (SEQ ID NO: 64) resembles round 2 RNA (Figure The saturability of binding is confirmed by the data in Figure 4; 90% of 5 nM 3 2 p-labeled F14.12 RNA binding is competed by excess cold RNA. Specificity is demonstrated by the results in Figure 5; binding of 5 nM 32 P-labeled F14.12 RNA is completely competed by the anti-L-selectin blocking monoclonal antibody, DREG-56, but is unaffected by an isotype-matched irrelevant antibody. These data validate the feasibility of using immobilized, purified protein to isolate ligands against a cell surface protein and the binding specificity of F14.12 to L-selectin in the context of a cell surface.

WO 96/40703 PCT/US96/09455 Example 11 Inhibition of Binding to Sialvl-Lewis x Oligonucleotide ligands, eluted by 2-5 mM EDTA, are expected to derive part of their binding energy from contacts with the lectin domain's bound Ca and consequently, are expected to compete with sialyl-Lewisx for binding. The ability of ligand F14.12 (SEQ ID NO: 78) to inhibit LS-Rg binding to immobilized sialyl- Lewis x was determined by competition ELISA assays. As expected, 4 mM EDTA reduced LS-Rg binding 7.4-fold, while 20 mM round 2 RNA did not inhibit LS-Rg binding. Carbohydrate binding is known to be Ca dependent; the affinity of round 2 RNA is too low to bind 10 nM LS-Rg (Table 7).

In this assay F14.12 RNA inhibits LS-Rg binding in a concentration dependent manner with an IC50 of about 10 nM (Figure Complete inhibition is observed at 50 nM F14.12. The observed inhibition is reasonable under the experimental conditions; the Kd of F14.12 at room temperature is about 1 nM (Table 9) and 10 nM LS-Rg is 20 nM binding sites. These data verify that RNA ligands compete with sialyl-Lewisx for LS-Rg binding and support the contention that low concentrations of EDTA specifically elute ligands that bind the lectin domain's carbohydrate binding site.

Example 12 Secondary Structure of High Affinity NH, Ligands to L-Selectin In favorable instances, comparative analysis of aligned sequences allows deduction of secondary structure and structure-function relationships. If the nucleotides at two positions in a sequence covary according to Watson-Crick base pairing rules, then the nucleotides at these positions are apt to be paired.

Nonconserved sequences, especially those that vary in length are not apt to be directly involved in function, while highly conserved sequence are likely to be directly involved.

Comparative analysis of the family I alignment suggests a hairpin structure in which the consensus sequence, AUGUGUGA, is contained within a variable size loop (Figure 7a). The stem sequences are not conserved and may be either 5' or 3'fixed or variable sequence. The one ligand that does not form a stem, F14.25 (SEQ ID NO: 73), has a significantly lower affinity than the other characterized ligands (Table 9).

The proposed structure for family Il is also a hairpin with the conserved sequence, AACAUGAAGUA, contained within a variable length loop (Figure 7b).

WO 96/40703 PCT/US96/09455 41 The 5'-half of the stem is 5'-fixed sequence which may account in part for the less highly conserved sequence, AGUC.

Although there is no alignment data for family II, the sequence folds into a pseudoknot (Figure 7c). Three attractive features of this model are 1) the helices stack on one another, 2) the structure utilizes only variable sequence and 3) the structure is compatible with the major variant sequences.

Example 13 ssDNA Ligands to Human L-Selectin The experimental procedures outlined in this Example were used to identify and characterize ssDNA ligands to human L-selectin as described in Examples 14-21.

Experimental Procedures A) Materials Unless otherwise indicated, all materials used in the ssDNA SELEX against the L-selectin/IgG2 chimera, LS-Rg, were identical to those of Example 7, paragraph A. The buffer for SELEX experiments was 1 mM CaCl2, 1 mM MgC12, 100 mM NaCI, 10.0 mM HEPES, pH 7.4. The buffer for all binding affinity experiments differed from the above in containing 125 mM NaCI, 5 mM KCI, and mM HEPES, pH 7.4.

B) SELEX The SELEX procedure is described in detail in United States Patent 5,270,163 and elsewhere. The strategy used for this ssDNA SELEX is essentially identical to that described in Example 7, paragraph B except as noted below. The nucleotide sequence of the synthetic DNA template for the LS-Rg SELEX was randomized at 40 positions. This variable region was flanked by BH 5' and 3' fixed regions. The random DNA template was termed 40BH (SEQ ID NO: 126) and had the following sequence: 5'-ctacctacgatctgactagc<40N>gcttactctcatgtagttcc-3'. The primers for the PCR were the following: 5' Primer: 5'-ctacctacgatctgactagc-3' (SEQ ID NO: 127) and 3' Primer: 5'-ajajaggaactacatgagagtaagc-3'; j=biotin (SEQ ID NO: 128). The fixed regions include primer annealing sites for PCR amplification. The initial DNA pool contained 500 pmols of each of two types of single-stranded DNA: 1) synthetic ssDNA and 2) PCR amplified, ssDNA from 1 nmol of synthetic ssDNA template.

For subsequent rounds, eluted DNA was the template for PCR amplification. PCR conditions were 50 mM KCI, 10 mM Tris-C1, pH 8.3, 7.5 mM WO 96/40703 PCT/US96/09455 42 MgC12, 1 mM of each dATP, dCTP, dGTP, and dTTP and 25 U/ml of the Stoffel fragment of Taq DNA polymerase. After PCR amplification, double stranded DNAs were end-labeled using 7 3 2 P-ATP. Complementary strands were separated by electrophoresis through an 8% polyacrylamide/7M urea gel. Strand separation results from the molecular weight difference of the strands due to biotintylation of the 3' PCR primer. In the final rounds, DNA strands were separated prior to end labelling in order to achieve high specific activity. Eluted fractions were processed by ethanol precipitation.

The strategy for partitioning LS-Rg/ssDNA complexes from unbound ssDNA was as described in Example 7, paragraph B, except that 2 mM EDTA was utilized for specific elution. The SELEX strategy is outlined in Table 11.

C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications and in Example 7, paragraph C, a nitrocellulose filter partitioning method was used to determine the affinity of ssDNA ligands for LS-Rg and for other proteins. For these experiments a Gibco BRL 96 well manifold was substituted for the 12 well Millipore manifold used in Example 7 and radioactivity was determined with a Fujix BAS100 phosphorimager.

Binding data were analyzed as described in Example 7, paragraph C.

D) Cloning and Sequencing Thirteenth, fifteenth and seventeenth round PCR products were re-amplified with primers which contain either a BamHI or a HinDI restriction endonuclease recognition site. Approximately 140 ligands were cloned and sequenced using the procedures described in Example 7, paragraph D. The resulting sequences are shown in Table 12.

E) Cell Binding Studies The ability of evolved ligand pools to bind to L-selectin presented in the context of a cell surface was tested in experiments with isolated human peripheral blood mononuclear cells (PBMCs) as described in Example 7, paragraph E Flow Cytometry Binding of oligonucleotides to leukocytes was tested in flow cytometry applications. Briefly, peripheral blood mononuclear cells (PBMC) were purified on histoplaque by standard techniques. Cells (500 cells/mL) were incubated with WO 96/40703 PCT/US96/09455 43 fluorescein labeled oligonucleotide in 0.25 mL SMHCK buffer (140 mM NaCI, 1 mM MgCl 2 1 mM CaCl,, 5 mM, KCI, 20 mM HEPES pH 7.4, 8.9 mM NaOH, 0.1% BSA, 0.1% sodium azide) at room temperature for 15 minutes.

Fluorescent staining of cells was quantified on a FACSCaliber fluorescent activated cell sorter (Becton Dickinson, San Jose, CA).

To examine the ability of oligonucleotides to bind leukocytes in whole blood, pl aliquots of heparinised whole blood were stained for 30 min at 220 C with 2 gg Cy5PE labeled anti-CD45 (generous gift of Ken Davis, Becton-Dickinson) and 0.15 gM FITC-LD201T1 (synthesized with a 5'-Fluorescein phosphoramidite by Operon Technologies, Alameda, CA; SEQ ID NO: 185). To determine specificity, other samples were stained with FITC-LD201T1 in the presence of 0.3 pM DREG- 56 or 7 p.M unlabeled LD201T1; or cells were reassayed after addition of 4 mM EDTA. The final concentration of whole blood was at least 70% Stained, concentrated whole blood was diluted 1/15 in 140 mM NaC1, 5 mM KC1, 1 mM MgC12, 1 mM CaC12, 20 mM HEPES pH 7.4, 0.1% bovine serum albumin and 0.1% NaN3 immediately prior to flow cytometry on a Becton-Dickinson FACS Calibur. Lymphocytes and granulocytes were gated using side scatter and staining.

F) Synthesis and Characterization of Multimeric Oligonucleotide Ligands Synthesis of Branched Dimeric Oligonucleotide Complexes Dimeric oligonucleotides were synthesized by standard solid state processes, with initiation from a Symmetric Linking CPG (Operon, Alameda, CA).

Branched complexes contain two copies of a truncated L-selectin DNA ligand, each of which is linked by the 3' end to the above CPG via a five unit ethylene glycol spacer (Figure 8A). Each ligand is labeled with a fluorescein phosphoramidite at the end (Glen Research, Sterling, VA). Branched dimers were made for 3 truncates of LD201T1 (SEQ ID NO: 142). The truncated ligands used were LD201T4 (SEQ ID NO: 187), LD201T10 (SEQ ID NO: 187) and LD201T1 (SEQ ID NO: 185).

Branched dimers were purified by gel electrophoresis.

Synthesis of Multivalent Biotintylated-DNA Ligand/Streptavidin Complexes Multivalent oligonucleotide complexes were produced by reacting biotintylated DNA ligands with either fluorescein or phycoerythrin labeled streptavidin (SA-FITC, SA-PE, respectively) (Figure 8B). Streptavidin (SA) is a tetrameric protein, each subunit of which has a biotin binding site. 5' and 3' biotintylated DNAs were synthesized by Operon Technologies, Inc (Alameda, CA) WO 96/40703 PCT/US96/09455 44 using BioTEG and BioTEG CPG (Glen Research, Sterling, VA), respectively. The expected stoichiometry is 2 to 4 DNA molecules per complex. SA/bio-DNA complexes were made for 3 truncates of LD201(SEQ ID NO: 142). The truncated ligands were LD201T4 (SEQ ID NO: 187), LD201T10 (SEQ ID NO: 188) and LD201T1 (SEQ ID NO: 185). The bio-DNA/SA multivalent complexes were generated by incubating biotin modified oligonucleotide (1 mM) and fluoroscein labeled streptavidin (0.17 mM) in 150 mM NaCI, 20 mM HEPES pH 7.4 at room temperature for at least 2 hours. Oligonucleotide-streptavidin complexes were used directly from the reaction mixture without additional purification of the Complex from free streptavidin or oligonucleotide.

Synthesis of a Dumbell Dimer Multivalent Complex A "dumbell" DNA dimer complex was formulated from a homobifunctional N-hydroxysuccinimidyl (or NHS) active ester of polyethelene glycol, PEG 3400 MW, and a 29mer DNA oligonucleotide, NX303 (SEQ ID NO: 196), having a terminal Amino Modifier C6 dT (Glen Research) and a terminal phosphodiester linkage (Figure 8C). NX303 is a truncate of LD201 (SEQ ID NO: 142). The conjugation reaction was in DMSO with 1% TEA with excess equivalents of the DNA ligand to PEG. The PEG conjugates were purified from the free oligonucleotide by reverse phase chromatography. The dimer was then purified from the monomer by anion exchange HPLC. The oligonucleotide was labeled at the 5' terminus with fluorescein as previously described.

Synthesis of a Fork Dimer Multivalent Complex To synthesize the fork dimer multivalent complex (Figure 8D), a glycerol was attached by its 2-position to one terminus of a linear PEG molecule (MW kD) to give the bis alcohol. This was further modified to the bis succinate ester, which was activated to the bis N-hydroxysuccinimidyl active ester. The active ester was conjugated to the primary amine at the 5' terminus of the truncated DNA nucleic acid ligand NX303 (SEQ ID NO: 196). The conjugation reaction was in DMSO with 1% TEA with excess equivalents of the DNA ligand to PEG. The PEG conjugates were purified away from the free oligonucleotide by reverse phase chromatography. The dimer was then purified away from the monomer by anion exchange HPLC. The oligonucleotide was labeled at the 5' terminus with fluorescein as previously described.

WO 96/40703 PCT/US96/09455 Characterization of Multimeric Oligonucleotide Ligands The binding of dimeric and multimeric oligonucleotide complexes to human peripheral blood mononuclear cells was analyzed by flow cytometry as described in Example 13, paragraph D.

G) Photo-Crosslinking A photo-crosslinking version of DNA ligand LD201T4 (SEQ ID NO: 187) was synthesized by replacing nucleotide T15 (Figure 12) with 4 nmol of 32 p-labeled DNA was incubated with 4 nmol L-selectin-Rg in 4 ml IX SHMCK 0.01 human serum albumin then irradiated at ambient temperature with 12,500 pulses from an excimer laser at a distance of 50 cm and at 175 mJ/pulse. Protein and DNA were precipitated with 400 pl 3 M sodium acetate and 8.4 ml ethanol followed by incubation at -70 degrees C. Precipitated material was centrifuged, vacuum dried and resuspended in 100 il 0.1 M Tris pH 8.0, mM CaCl2. Fourty-five gig chymotrypsin were added and after 20 min at 37 degrees C, the material was loaded onto an 8% polyacrylamide/7 M urea/ 1XTBE gel and electrophoresed until the xylene cyanole had migrated 15 cm. The gel was soaked for 5 min in 1X TBE and then blotted for 30 min at 200 mAmp in 1XTBE onto Immobilon-P (Millipore). The membrane was washed for 2 min in water, air dried, and an autoradiograph taken. A labeled band running slower than the free DNA band, representing a chymotryptic peptide crosslinked to LD201T4, was observed and the autoradiograph was used as a template to excise this band from the membrane. The peptide was sequenced by Edman degradation, and the resulting sequence was LEKTLP_SRSYY. The blank residue corresponds to the crosslinked amino acid, F82 of the lectin domain.

H) Lymphocyte Trafficking Experiments Human PBMC were purified from heparinised blood by a Ficoll-Hypaque gradient, washed twice with HBSS (calcium/magnesium free) and labeled with 5 1 Cr (Amersham). After labeling, the cells were washed twice with HBSS (containing calcium and magnesium) and 1% bovine serum albumin (Sigma). Female SCID mice (6-12 weeks of age) were injected intravenously with 2x10 6 cells. The cells were either untreated or mixed with either 13 pmol of antibody (DREG-56 or MEL- 14), or 4, 1, or 0.4 nmol of modified oligonucleotide (synthesis described below).

One hour later the animals were anesthetized, a blood sample taken and the mice were euthanised. PLN, MLN, Peyer's patches, spleen, liver, lungs, thymus, WO 96/40703 PCT/US96/09455 46 kidneys and bone marrow were removed and the counts incorporated into the organs determined by a Packard gamma counter. In a second protocol, 2x10 6 human PBMC, purified, labeled, and washed as described above, were injected intravenously into female SCID mice without antibody or oligonucleotide pretreatment. One to 5 min prior to injecting the cells, the animals were injected with either 15 pmol DREG-56 or 4 nmol modified oligonucleotide. Counts incorporated into organs were quantified as described above.

Synthesis of modified nucleotides NX288 (SEQ ID NO: 193) and NX303 (SEQ ID NO: 196) was initiated by coupling to a dT-5'-CE polystyrene support (Glen Research), resulting in a terminal phosphodiester linkage, and having a 5' terminal an Amino Modifier C6 dT (Glen Research). Once NX288 and NX303 were synthesized, a 20,000 MW PEG2-NHS ester (Shearwater Polymers, Huntsville, AL) was then coupled to the oligonucleotide through the 5' amine moiety. The molar ratio, PEG:oligo, in the reactions was from 3:1 to 10:1. The reactions were performed in 80:20 100 mM borate buffer pH 8: DMF at 370 C for one hour.

I) Inhibition of L-selectin Binding to Sialyl Lewisx SLeX-BSA (Oxford GlycoSystems, Oxford, UK) in IX PBS, without CaC12 and MgC12,'(GIBCO/BRL) was immobilized at 100 ng/well onto a microtiter plate by overnight incubation at 220 C. The wells were blocked for 1 h with the assay buffer consisting of 20 mM HEPES, 111 mM NaCI, 1 mM CaC12, 1 mM MgCl2, 5 mM KC1, 8.9 mM NaOH, final pH 8, and 1% globulin-free BSA (Sigma). The reaction mixtures, incubated for 90 min with orbital shaking, contained 5 nM L-Selectin-Rg, a 1:100 dilution of anti-human IgG-peroxidase conjugate (Sigma), and 0 50 nM of competitor in assay buffer. After incubation, the plate was washed with BSA-free assay buffer to remove unbound chimera-antibody complex and incubated for 25 min with Ophenylenediamine dihydrochloride peroxidase substrate (Sigma) by shaking in the dark at 220 C. Absorbance was read at 450 nm on a Bio-Kinetics Reader, Model EL312e (Bio-Tek Instruments, Laguna Hills, CA). Values shown represent the mean s.e from duplicate, or triplicate, samples from one representative experiment.

WO 96/40703 PCTIUS96/09455 47 Example 14 ssDNA Ligands to L-Selectin A. SELEX The starting ssDNA pool for SELEX, randomized 40BH (SEQ ID NO: 126), contained approximately 1015 molecules (1 nmol ssDNA). The dissociation constant of randomized ssDNA to LS-Rg is estimated to be approximately 10 gM.

The SELEX protocol is outlined in Table 11.

The initial round of SELEX was performed at 4 °C with an LS-Rg density of 16.7 pmol/l of protein A sepharose beads. Subsequent rounds were at room temperature except as noted in Table 11. The 2 mM EDTA elution was omitted from rounds 1-3. The signal to noise ratio of the 50 mM EDTA elution in these three rounds was 50, 12 and 25, respectively (Table 11). These DNAs were amplified for the input materials of rounds 2-4. Beginning with round 4, a 2 mM EDTA elution was added to the protocol. In this and all subsequent rounds, the 2 mM EDTA eluted DNA was amplified for the next round's input material.

To increase the stringency of selection, the density of immobilized LS-Rg was reduced ten fold in round 4 with further reductions in protein as needed to increase the stringency of selectin (Table 11). Under these conditions a rapid increase in the affinity of the selected pools was observed (Tables 11); at 4 OC, the dissociation constant of round 7 ssDNA was 60 nM.

Binding experiments with 7th round DNA revealed that the affinity of the evolving pool for L-selectin was weakly temperature sensitive (Kds: 60 nM, 94 nM and 230 nM at 4 room temperature and 37 respectively). To enhance the selection of ligands that bind at physiological temperature, rounds 8, 13, 16 and 17 were performed at 37 Although temperature sensitive, the affinity of round ssDNA was optimal at room temperature (160 pM), with 3-fold higher Kds at 4 °C and 37 °C.

Bulk sequencing of DNA pools indicates some non-randomness at round and dramatic non-randomness at round 13. Ligands were cloned and sequenced from rounds 13, 15, and 17. Sequences were aligned manually and with the aid of a NeXstar computer program that determines consensus sequences from frequently occurring local alignments.

B. Sequences In Table 12, ligand sequences are shown in standard single letter code (Comish-Bowden, 1985 NAR 13: 3021-3030). Only the evolved random region is shown in Table 12. Any portion of the fixed region is shown in lower case letters.

WO 96/40703 PCTIUS96/09455 48 By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise A unique sequence is operationally defined as one that differs from all others by three or more nucleotides.

Sequences that were isolated more than once are indicated by the parenthetical number, following the ligand isolate number. These clones fall into six families and a group of unrelated sequences or orphans (Table 12)(SEQ ID NOs: 129-180).

Family 1 is defined by ligands from 33 lineages and has a well defined consensus sequence, TACAAGGYGYTAVACGTA (SEQ ID NO: 181). The conservation of the CAAGG and ACG and their 6 nucleotide spacing is nearly absolute (Table 12). The consensus sequence is flanked by variable but complementary sequences that are 3 to 5 nucleotides in length. The statistical dominance of family 1 suggests that the properties of the bulk population are a reflection of those of family 1 ligands. Note that ssDNA family I and 2'-NH2 family I share a common sequence, CAAGGCG and CAAGGYG, respectively.

Family 2 is represented by a single sequence and is related to family 1. The ligand contains the absolutely conserved CAAGG and highly conserved ACG of family 1 although the spacing between the two elements is strikingly different (23 compared to 6 nucleotides).

Families 4-6 are each defined by a small number of ligands which limits confidence in their consensus sequence, while family 7 is defined by a single sequence which precludes determination of a consensus. Family 5 appears to contain two conserved sequences, AGGGT and RCACGAYACA, the positions of which are circularly permuted.

C. Affinities The dissociation constants of representative ligands from Table 12 are shown in Table 13. These calculations assume two ssDNA ligand binding sites per chimera. The affinity of random ssDNA cannot be reliably determined but is estimated to be approximately 10 pLM.

At room temperature, the dissociation constants range from 43 pM to 1.8 nM which is at least a 5x10 3 to 2x10 5 fold improvement over randomized ssDNA (Table 13). At 37 OC, the Kds range from 130 pM to 23 nM. The extent of temperature sensitivity varies from insensitive (ligands LD122 and LD127 (SEQ ID NO: 159 and 162)) to 80-fold (ligand LD112 (SEQ ID NO: 135)). In general, among family 1 ligands the affinity of those from round 15 is greater than that of those from round 13. For the best ligands (LD208, LD227, LD230 and LD233 WO 96/40703 PCTIUS96/09455 49 (SEQ ID NOS: 133, 134, 132, and 146)), the difference in affinity at room temperature and 37 0 C is about 4-fold.

The observed affinities of the evolved ssDNA ligand pools reaffirm our proposition that it is possible to isolate oligonucleotide ligands with affinities that are several orders of magnitude greater than that of carbohydrate ligands.

Example Specificity of ssDNA Ligands to L-Selectin The affinity of representative cloned ligands for LS-Rg, ES-Rg, PS-Rg, CD22P-Rg and WGA was determined by nitrocellulose partitioning and the results shown in Table 14. The ligands are highly specific for L-selectin. The affinity for ES-Rg is about 10 3 -fold lower and that for PS-Rg is about 5x10 3 -fold less than for LS-Rg. Binding above background is not observed for CD22P-Rg or for WGA at 0.7 and 1.4 p.M protein, respectively, indicating that ligands neither bind the Fc domain of the chimeric constructs nor have affinity for unrelated sialic acid binding sites.

The specificity of oligonucleotide ligand binding contrasts sharply with the binding of cognate carbohydrates by the selectins and reconfirms the proposition that SELEX ligands will have greater specificity than carbohydrate ligands.

Example 16 Cell Binding Studies Round 15 ssDNA pool was tested for its ability to bind to L-selectin presented in the context of a peripheral blood mononuclear cell surface as described in Example 13, paragraph E. The evolved pool was tested both for affinity and for specificity by competition with an anti-L-selectin monoclonal antibody. Figure 9 shows that the round 15 ssDNA pool binds isolated PBMCs with a dissociation constant of approximately 1.6 nM and, as is expected for specific binding, in a saturable fashion. Figure 10 directly demonstrates specificity of binding; in this experiment, binding of 2 nM 3 2 P-labeled round 15 ssDNA is completely competed by the anti-L-selectin blocking monoclonal antibody, DREG-56, but is unaffected by an isotype-matched irrelevant antibody. In analogous experiments, LD201T1 (SEQ ID NO: 185) was shown to bind human PBMC with high affinity. Binding was saturable, divalent cation dependent, and blocked by DREG-56.

These data validate the feasibility of using immobilized, purified protein to isolate ligands against a cell surface protein and demonstrate the specific binding of WO 96/40703 PCT/US96/09455 the round 15 ssDNA pool and of ligand LD201T1 to L-selectin in the context of a cell surface.

The binding of LD201T1 to leukocytes in whole blood was examined by flow cytometry. Fluorescein isothiocyanate (FITC)-conjugated LD201T1 specifically bind human lymphocytes and neutrophils (Figure 11A/B); binding is inhibited by competition with DREG-56, unlabeled LD201, and by the addition of 4 mM EDTA (Figure 11A/B). These cell binding studies demonstrate that LD201T1 bind saturably and specifically to human L-selectin on lymphocytes and neutrophils.

Example 17 Secondary Structure of High Affinity ssDNA Ligands to L-Selectin In favorable instances, comparative analysis of aligned sequences allows deduction of secondary structure and structure-function relationships. If the nucleotides at two positions in a sequence covary according to Watson-Crick base pairing rules, then the nucleotides at these positions are apt to be paired.

Nonconserved sequences, especially those that vary in length are not apt to be directly involved in function, while highly conserved sequence are likely to be directly involved.

Comparative analysis of 24 sequences from family 1 strongly supports a hairpin secondary structure for these ligands (Figure 12). In the figure, consensus nucleotides are specified, with invariant nucleotides in bold type. To the right of the stem is a matrix showing the number of occurrences of particular base pairs for the positions in the stem that are on the same line. The deduced structure consists of a GYTA tetraloop, a 3 nucleotide-pair upper stem and a 6 to 7 nucleotide-pair lower stem. The upper and lower stems are separated by an asymmetrical, AA internal loop or "bulge." Two of the three base pairs in the upper stem and 6 of 7 in the lower stem are validated by covariation. The two invariant pairs, positions 7/20 and 10/19 are both standard Watson/Crick basepairs. This structure provides a plausible basis for the direct involvement of invariant nucleotides (especially, A8, A9 and in binding the target protein.

The site of oligonucleotide binding on L-selectin can be deduced from a set of competition experiments. DREG56 is an anti-L-selectin, adhesion blocking monoclonal antibody that is known to bind to the lectin domain. Binding of three unrelated ligands, LD201T1 (SEQ ID NO: 185), LD174T1 (SEQ ID NO: 194) and LD196T1 (SEQ ID NO: 195), to LS-Rg was blocked by DREG-56, but not by an isotype-matched control. In cross-competition experiments, LD201T1, LD174T1, or LD196T1 prevented radio-labeled LD201T1 from binding to LS-Rg, consistent WO 96/40703 PCT/US96/09455 51 with the premise that the ligands bind the same or overlapping sites. The blocking and competition experiments, taken together with divalent cation-dependence of binding, suggest that all three ligands bind to the lectin domain. This conclusion has been verified for LD201 by photo-crosslinking experiments.

If T15 of LD201T4 (SEQ ID NO: 187; Figure 12) is replaced with uracil, the resulting DNA photo-crosslinks at high yield to LS-Rg following irradiation with an excimer laser as described in Example 13, paragraph G. The high yield of crosslinking indicates a point contact between the protein and Sequencing of the chymotryptic peptide corresponding to this point contact revealed a peptide deriving from the lectin domain; F82 is the crosslinking amino acid. Thus, F82 contacts T15 in a stacking arrangement that permits high yield photocrosslinking. By analogy to the structure of the highly related E-selectin (Graves et al, Nature 367, 532-538, 1994), F82 is adjacent to the proposed carbohydrate binding site. Thus, this photo-crosslink provides direct evidence that ligand LD201 makes contact with the lectin domain of LS-Rg and provides an explanation for the function of the oligonucleotides in either sterically hindering access to the carbohydrate binding site or in altering the conformation of the lectin domain upon DNA binding.

Example 18 L-Selectin ssDNA Ligand Truncate Data Initial experiments to define the minimal high affinity sequence of family 1 ligands show that more than the 26 nucleotide hairpin (Figure 12; Table 13) is required. Ligands corresponding to the hairpin, LD201T4 (SEQ ID NO: 187) and LD227T1 (SEQ ID NO: 192) derived from LD201 (SEQ ID NO: 173) and LD227 (SEQ ID NO: 134), respectively, bind with 20-fold and 100-fold lower affinity than their full length progenitors. The affinity ofLD201T3 (SEQ ID NO: 186), a 41 nucleotide truncate of ligand LD201, is reduced about 15-fold compared to the full length ligand, while the affinity of the 49-mer LD201T1 (SEQ ID NO: 185) is not significantly altered (Tables 12 and 13).

Additional experiments show that truncates LD201T10 (SEQ ID NO: 188) and LD227X1 (SEQ ID NO: 191) bind with affinities similar to their full length counterparts. Both of these ligands have stems that are extended at the base of the consensus stem. Alterations in the sequence of the added stem have little, if any, effect on binding, suggesting that it is not directly involved in binding The added stem is separated from the consensus stem by a single stranded bulge. The two ligands' single stranded bulges differ in length and have unrelated WO 96/40703 PCT/US96/09455 52 sequences. Furthermore, LD201's bulge is at the 5'-end of the original stem base while that of LD227 is at the 3'-end. Thus, the two ligands do not present an obvious consensus structure. Removal of the loop (LD201) or scrambling or truncating the sequence (LD227) diminishes affinity, suggesting that the bulged sequences may be directly involved in binding. Note that although LD201T3 is longer than LD201T10, it is unable to form the single stranded loop and extended stem because of the position of the truncated ends.

Example 19 Inhibition of Binding to Sialvl Lewisx Sialyl Lewisx is the minimal carbohydrate ligand bound by selectins. The ability of ssDNA ligands to inhibit the binding of L-selectin to Sialyl Lewis x was determined in competition ELISA assays as described in Example 13, paragraph I.

LD201T1 (SEQ ID NO: 185), LD174T1 (SEQ ID NO: 194) and LD196T1 (SEQ ID NO: 195) inhibited LS-Rg binding to immobilized SLex in a dose dependent manner with IC50s of approximately 3 nM. This is a 10 5 -10 6 -fold improvement over the published IC50 values for SLe x in similar plate-binding assays. A scrambled sequence based on LD201T1 showed no activity in this assay. These data verify that DNA ligands compete with sialyl-Lewisx for LS-Rg binding and support the contention that low concentrations of EDTA specifically elute ligands that bind the lectin domain's carbohydrate binding site.

Example Inhibition Of Lymphocte Trafficking by L-Selectin ssDNA Ligands Lymphocyte trafficking to peripheral lymph nodes is exquisitely dependent on L-selectin. Since the ssDNA ligands binds to human but not rodent L-selectin, a xenogeneic lymphocyte trafficking system was established to evaluate in vivo efficacy. Human PBMC, labeled with 5 1 Cr, were injected intravenously into SCID mice. Cell trafficking was determined 1 hour later. In this system, human cells traffic to peripheral and mesenteric lymph nodes (PLN and MLN). This accumulation is inhibited by DREG-56 (Figure 13) but not MEL-14, a monoclonal antibody that blocks murine L-selectin-dependent trafficking. In initial experiments cells were incubated with either DREG-56 or 3' capped and PEG-modified oligonucleotide before injection. NX288 (SEQ ID NO: 193) inhibited trafficking of cells to PLN (Figure 13) and MLN in a dose-dependent fashion but had no effect on the accumulation of cells in other organs. At the highest dose tested (4 nmol), WO 96/40703 PCT/US96/09455 53 inhibition by the DNA ligand was comparable to that of DREG-56 (13 pmol), while a scrambled sequence had no significant effect (Figure 13). The activity of LD174T1 (SEQ ID NO: 194) was similar to that of NX288.

To determine if the modified oligonucleotide was effective when it was not pre-incubated with cells, DREG-56 (13 pmol/mouse) or the modified oligonucleotide (4 nmol/mouse) was injected intravenously into animals and 1-5 min later the radio-labeled human cells were given intravenously. Again, both NX288 (SEQ ID NO: 193) and DREG-56 inhibited trafficking to PLN and MLN while the scrambled sequence had no effect (Figure 14). Therefore, the modified oligonucleotide did not require pre-incubation with the cells to effectively block trafficking. These experiments demonstrate, in vivo, the efficacy of oligonucleotide ligands in inhibiting a L-selectin dependent process.

Example 21 L-Selectin Nucleic Acid Ligand Multimers Multivalent Complexes were made in which two nucleic acid ligands to Lselectin were conjugated together. Multivalent Complexes of nucleic acid ligands are described in copending United States Patent Application Serial Number 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes" which is herein incorporated by reference in its entirety. These multivalent Complexes were intended to increase the binding energy to facilitate better binding affinities through slower off-rates of the nucleic acid ligands. These multivalent Complexes may be useful at lower doses than their monomeric counterparts. In addition, high molecular weight (20kD) polyethylene gylcol (PEG) was included in some of the Complexes to decrease the in vivo clearance rate of the complexes. Specifically, the nucleic acid ligands incorporated into the Complexes were LD201T1 (SEQ ID NO: 185), LD201T4 (SEQ ID NO: 187), LD201T10 (SEQ ID NO: 188) and NX303 (SEQ ID NO: 196). Multivalent selectin nucleic acid ligand Complexes were produced as described in Example 13, paragraph F.

A variety of monomeric nucleic acid ligands and multivalent Complexes have been examined in flow cytometry. The multivalent Complexes exhibited similar specificity to the monomeric forms, but enhanced affinity as well as improved slower) off-rate for human lymphocytes. Titration curves, obtained from incubating fluorescently labeled monomeric FITC-LD201T1 with peripheral blood mononuclear cells (PBMC) purified human lymphocytes, indicated that binding to cells is saturable. Half-saturation fluorescence occurred at 3 nM oligonucleotide. In contrast, the branched dimeric FITC-LD201T1 and bio-LD201T1/SA multivalent WO 96/40703 PCT/US96/09455 54 Complexes exhibited half-saturation at approximately 0.15 nM, corresponding to an apparent 20-fold increase in affinity. In similar experiments, half saturation of the dumbell and fork dimers of LD201T4 was observed at 0.1 and 0.6 nM, respectively, compared to 20 nM for monomeric LD201T4.

Kinetic competition experiments were performed on monomeric nucleic acid ligands and multivalent Complexes. Kinetic competition experiments were performed with PBMC purified lymphocytes. Cells were stained as described above but used 10 nM oligonucleotide. The off-rate for monomeric, dimeric and multivalent Complexes was determined by addition of 500 nM unlabeled oligonucleotide to cells stained with fluorescently labeled ligand and measurement of the change in the mean fluorescence intensity as a function of time. The dissociation rate of a monomeric LD201T1 from L-selectin expressing human lymphocytes was approximately 0.005 sec-1, corresponding to a half-life of roughly 2.4 minutes.

The LD201T1 branched dimer and biotin conjugate multivalent Complexes exhibited apparent off-rates several times slower than that observed for the monomeric ligand and as slow or slower than that observed for the anti-L-selectin blocking antibody DREG56, determined under the same conditions. A multivalent Complex containing a non-binding nucleic acid sequence did not stain cells under identical conditions and did not compete in the off-rate experiments. The off-rate of the LD201T4 dumbell and fork dimers is faster than the LD201T1 branched dimer and is better than all monomers tested. These results confirm the proposition that dimeric and multimeric ligands bind with higher affinities than do monomeric ligands and that the increased affinity results from slower off-rates.

Example 22 2'-F RNA Ligands to Human L-Selectin The experimental procedures outlined in this Example were used to identify and characterize 2'-F RNA ligands to human L-selectin as described in Examples 23-25.

Experimental Procedures A) Materials Unless otherwise indicated, all materials used in the 2'-F RNA SELEX against the L-selectin/IgG2 chimera, LS-Rg, were identical to those of Examples 7, paragraph A and 13, paragraph A. SHMCK-140 buffer, used for all SELEX and binding experiments, was 1 mM CaC12, 1 mM MgC12, 140 mM NaCI, 5 mM KC1, and 20 mM HEPES, pH 7.4. A soluble form of L-selectin, corresponding to the WO 96/40703 PCTfUS96/09455 extracellular domains, was purchased from R&D Systems and used for some nitrocellulose filter binding experiments.

B) SELEX The SELEX procedure is described in detail in United States Patent 5,270,163 and elsewhere. Procedures are essentially identical to those in Examples 7 and 13 except as noted. The variable regions of synthetic DNA templates were randomized at either 30 or 40 positions and were flanked by N7 5' and 3' fixed regions producing transcripts 30N7 (SEQ ID NO: 292) and 40N7 (SEQ ID NO: 389). The primers for the PCR were the following: N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3' (SEQ ID NO: N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66) The initial RNA pool was made by first Klenow extending 3 nmol of synthetic single stranded DNA and then transcribing the resulting double stranded molecules with T7 RNA polymerase. Klenow extension conditions: 6 nmols primer 5N7, 3 nmols 30N7 or 40n7, IX Klenow Buffer, 1.8 mM each of dATP, dCTP, dGTP and dTTP in a reaction volume of 0.5 ml.

For subsequent rounds, eluted RNA was the template for AMV reverse transcriptase mediated synthesis of single-stranded cDNA. These single-stranded DNA molecules were converted into double-stranded transcription templates by PCR amplification. PCR conditions were 50 mM KC1, 10 mM Tris-C1, pH 8.3, 7.5 mM MgCl2, 0.2 mM of each dATP, dCTP, dGTP, and dTTP, and 100 U/ml of Taq DNA polymerase. Transcription reactions contained one third of the purified PCR reaction, 200 nM T7 RNA polymerase, 80 mM HEPES (pH 12 mM MgCl2, mM DTT, 2 mM spermidine, 1 mM each of 2'-OH ATP, 2'-OH GTP, 3 mM each of 2'-F CTP, 2'-F UTP, and 250 nM a- 3 2 P 2'-OH ATP. Note that in all transcription reactions 2'-F CTP and 2'-F UTP replaced CTP and UTP.

The strategy for partitioning LS-Rg/RNA complexes from unbound RNA is outlined in Table 15 and is essentially identical to that of Example 7, paragraph B.

In the initial SELEX rounds, which were performed at 37 oC, the density of immobilized LS-Rg was 10 pmols/pl of Protein A Sepharose 4 Fast Flow beads.

LS-Rg was coupled to protein A sepharose beads according to the manufacturer's instructions (Pharmacia Biotech). In later rounds, the density of LS-Rg was reduced (Table 15), as needed, to increase the stringency of selection. At the seventh round, both SELEXes were branched. One branch was continued as previously described (Example 7, paragraph In the second branch of both WO 96/40703 PCT/S96/09455 56 SELEXes, the RNA pool was pre-annealed to oligonucleotides that are complementary to the 5' and 3' fixed sequences. These rounds are termed "counterselected" rounds. Before each round, RNA was batch adsorbed to 100 Rl of protein A sepharose beads for 15 minutes in a 2 ml siliconized column. Unbound RNA and RNA eluted with minimal washing (two volumes) were combined and used for SELEX input material. For SELEX, extensively washed, immobilized LS-Rg was batch incubated with pre-adsorbed RNA for 1 to 2 hours in a 2 ml column with constant rocking. Unbound RNA was removed by extensive batch washing (500 pl SHMCK 140/wash). In addition, the counter selected rounds were extensively washed with buffer containing 200 nM of both complementary oligos. Bound RNA was eluted as two fractions; first, bound RNA was eluted by incubating and washing columns with 100 gL 5 mM EDTA in SHMCK 140 without divalent cations; second, the remaining elutable RNA was removed by incubating and/or washing with 500 pL 50 mM EDTA in SHMCK 140 without divalents. The percentage of input RNA that was eluted is recorded in Table 22. In every round, an equal volume of protein A sepharose beads without LS-Rg was treated identically to the SELEX beads to determine background binding. All unadsorbed, wash and eluted fractions were counted in a Beckman LS6500 scintillation counter in order to monitor each round of SELEX.

The 5 mM EDTA eluates were processed for use in the following round (Table 15). After precipitating with isopropanol/ethanol the RNA was reverse transcribed into cDNA by AMV reverse transcriptase either at 48 °C for minutes and then 65 °C for 15 minutes in 50 mM Tris-C1 pH 60 mM NaC1, 6 mM Mg(OAc)2, 10 mM DTT, 200 pmol DNA primer, 0.5 mM each of dNTPs, and 0.4 unit/pL AMV RT. Transcripts of the PCR product were used to initiate the next round of SELEX.

C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to determine the affinity of RNA ligands for LS-Rg and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 p.m pore size, Millipore) were placed on a vacuum manifold and washed with 3 ml of SHMCK 140 buffer under vacuum. Reaction mixtures, containing 3 2 p labeled RNA pools and unlabeled LS-Rg, were incubated in SHMCK 140 for 10 min at 37 and then immediately washed with 3 ml SHMCK 140. The filters were air-dried and counted in a Beckman LS6500 liquid scintillation counter without WO 96/40703 PCT/US96/09455 57 fluor. Alternatively, binding studies employed 96 well micro-titer manifolds essentially as described in Example 13, paragraph E.

D) Cloning and Sequencing 12th round PCR products were re-amplified with primers which contain either a BamHI or a HinDmI restriction endonuclease recognition site. Using these restriction sites, the DNA sequences were inserted directionally into the pUC9 vector. These recombinant plasmids were transformed into E. coli strain DH5a (Life Technologies, Gaithersburg, MD). Plasmid DNA was prepared according to the alkaline lysis method (Quiagen, QIAwell, Chattsworth CA). Approximately 300 clones were sequenced using the ABI Prism protocol (Perkin Elmer, Foster City, CA). Sequences are shown in Table 16.

E) Cell Binding Studies Binding of evolved ligands to L-selectin presented in the context of a cell surface was tested by flow cytometry experiments with human lymphocytes.

Briefly, peripheral blood mononuclear cells (PBMC) were purified on histoplaque by standard techniques. To evaluate leukocyte binding by unlabeled 2'-F ligands, cells (500 cells/mL) were incubated with fluorescein labeled FITC-LD201T1

(SEQ

ID NO: 185) in the presence of increasing concentrations of individual, unlabeled 2'- F ligands in 0.25 mL SMHCK buffer (140 mM NaCI, 1 mM MgCl 2 1 mM CaC1 2 mM, KC1, 20 mM HEPES pH 7.4, 8.9 mM NaOH, 0.1% BSA, 0.1% sodium azide) at room temperature for 15 minutes. Fluorescent staining of cells was quantified on a FACSCaliber fluorescent activated cell sorter (Becton Dickinson, San Jose, CA). The affinity of the 2'-F competitor was calculated from the flurorescence inhibition curves.

Example 23 2'-F RNA Ligands to L-Selectin A. SELEX The starting RNA pools for SELEX, randomized 30N7 (SEQ ID NO: 292) or 40N7 (SEQ ID NO: 389) contained approximately 1014 molecules (0.7 nmol RNA). The SELEX protocol is outlined in Table 15 and Example 22. All rounds were selected at 37 0 C. The dissociation constant of randomized RNA to LS-Rg is estimated to be approximately 10 pM. After six rounds the pool affinities had improved to approximately 300 nM. An aliquot of the RNA recovered from the seventh round was used as the starting material for the first counter-selected rounds.

WO 96/40703 PCT/US96/09455 58 Five rounds of counter-selection and five additional standard rounds were performed in parallel. Thus, a total of twelve rounds were performed in both branches of both SELEXes: 30N7, counter-selected 30N7, 40N7 and counter-selected 40N7. The affinities of each of the 12th round pools ranged from 60 to 400 pM. Ligands were cloned from these pools.

B. Sequences of 2'-F RNA Ligands to L-Selectin In Table 16, ligand sequences are shown in standard single letter code (Corish-Bowden, 1985 NAR 13: 3021-3030). Fixed region sequence is shown in lower case letters. By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides. Sequences that were isolated more than once are indicated by the parenthetical number, following the ligand isolate number.

The 30N7 and 40N7 SELEX final pools shared a common major sequence family, even though identical sequences from the two SELEXes are rare (Table 16).

Most ligands (72 of the 92 unique sequences) from the 30N7 and 40N7 SELEXes contain one of two related sequence motifs, RYGYGUUUUCRAGY or RYGYGUUWWUCRAGY. These motifs define family 1. Within the family there are three subfamilies. Subfamily la ligands (53/66) contain an additional sequence motif, CUYARRY, one nucleotide 5' to the family 1 consensus motifs. Subfamily lb (9/66 unique sequences) lacks the CUYARRY motif. Subfamily Ic (5/66) is also missing the CUYARRY motif, has an A inserted between the Y and G of consensus YGUU and lacks the consensus GA base pair. The significance of the sequence subfamilies is reflected in the postulated secondary structure of the ligands (Example A second family, composed of 5 sequences, has a relatively well defined consensus: UACUANo,_UGURCG...UYCACUAAGNi-2CCC (Table 16). Family 3 has a short, unreliable consensus motif (Table 16). In addition, there are approximately 12 orphans or apparently unrelated sequences. Three of the orphan sequences were recovered at least twice (Table 16).

C. Affinities The dissociation constants of representative ligands from Table 16 are shown in Table 17. These calculations assume two ligand binding sites per chimera.

WO 96/40703 PCT/US96/09455 59 The affinity of random 2'-F RNA cannot be reliably determined but is estimated to be approximately 10 pM.

The dissociation constants range from 34 pM to 315 nM at 37 Binding affinity is not expected to be temperature sensitive since selection was at 37 0 C and 2'-F RNA forms thermal stable structures, but binding has not been tested at lower temperatures. For the most part, the extreme differences in affinity may be related to predicted secondary structure (Example The observed affinities of the evolved 2'-F RNA ligands reaffirm our proposition that it is possible to isolate oligonucleotide ligands with affinities that are several orders of magnitude greater than that of carbohydrate ligands.

Example 24 Cell Binding Studies The ability of full length 2'-F ligands to bind to L-selectin presented in the context of a cell surface was tested by competition-flow cytometry experiments with human peripheral blood lymphocytes. Lymphocytes were stained with 10 nM FITC-conjugated DNA ligand FITC-LD201T1 (SEQ ID NO: 185) in the presence of increasing concentrations of unlabeled 2'-F ligands as described in Example 22, paragraph E. Ligands LF1513 (SEQ ID NO: 321), LF1514 (SEQ ID NO: 297), LF1613 (SEQ ID NO: 331) and LF1618 (SEQ ID NO: 351) inhibited the binding of FITC-LD201T1 in a concentration dependent manner, with complete inhibition observed at competitor concentrations of 10 to 300 nM. These results demonstrate that the 2'-F ligands are capable of binding cell surface L-selectin and suggest that the 2'-F ligands and LD201T1 bind the same or overlapping sites. The affinities of the fluoro ligands, calculated from the competition curves, range from 0.2 to 25 nM.

The affinity of two of the ligands for L-selectin on human lymphocytes, LF1613 (Kd 0.2 nM) and LF1514 (Kd 0.8 nM), is significantly better than that of the DNA ligand LD201T1 (Kd 3 nM). The reasonable agreement between the affinities for purified protein and lymphocyte L-selectin suggests that binding to lymphocytes is specific for L-selectin. These data validate the feasibility of using immobilized, purified protein to isolate ligands against a cell surface protein.

Example Secondary Structure of High Affinity 2'-F RNA Ligands to L-Selectin In favorable instances, comparative analysis of aligned sequences allows deduction of secondary structure and structure-function relationships. If the nucleotides at two positions in a sequence covary according to Watson-Crick base WO 96/40703 PCTIUS96/09455 pairing rules, then the nucleotides at these positions are apt to be paired.

Nonconserved sequences, especially those that vary in length are not apt to be directly involved in function, while highly conserved sequence are likely to be directly involved.

The deduced secondary structure of family la ligands from comparative analysis of 21 unique sequences is a hairpin motif (Figure 15) consisting of a 4 to 7 nucleotide terminal loop, a 6 base upper stem and a lower stem of 4 or more base pairs. The consensus terminal loops are either a UUUU tetraloop or a UUWWU pentaloop. Hexa- and heptaloops are relatively rare. The upper and lower stems are delineated by a 7 nucleotide bulge in the 5'-half of the stem. Four of the six base pairs in the upper stem and all base pairs in the lower stem are supported by Watson- Crick covariation. Of the two invariant base pairs in the upper stem, one is the loop closing GC, while the other is a non-standard GA. The lower stem is most often 4 or 5 base pairs long but can be extended. While the sequence of the upper stem is strongly conserved, that of the lower stem is not, with the possible exception of the YR' base pair adjacent to the internal bulge. This base pair appears to covary with the 3' position of the 7 nucleotide bulge in a manner which minimizes the likelihood of extending the upper stem. Both the sequence (CUYARRY) and length (7 nt) of the bulge are highly conserved.

In terms of comparative analysis, the 7 nucleotide bulge, the upper stem and the 5' and 3' positions of the terminal loop are most apt to be directly involved in Lselectin binding. Specifically, the 5' U and 3' U of the terminal loop, the invariant GC and GA base pairs of the upper stem and the conserved C, U and A of the bulge are the mostly likely candidates. The lower stem, because of its variability in length and sequence, is less likely to be directly involved. The importance of the bulge for binding is supported by the poor affinity of ligand LF1512 (SEQ ID NO: 357; Kd 315 nM); the simplest structure for this ligand is a UUUU tetraloop and a ten base pair, nearly perfect, consensus stem which is missing only the 7 nucleotide bulge.

The deduced secondary structure of family lb is similar to that of family la, except that the upper stem is usually 7 base pairs in length and that the single stranded bulge which does not have a highly conserved consensus is only 4 nucleotide long. This structure may be an acceptable variation of the la secondary structure with the upper stem's increased length allowing a shorter bulge; the affinity of ligand LF1511 (SEQ ID NO: 332) is 300 pM.

Although family lc has a consensus sequence, GUUUUCNR that is related to la and lb, a convincing consensus secondary structure is not evident, perhaps due to insufficient data. The most highly structured member of the family, LF1618 WO 96/40703 PCT/US96/09455 61 (SEQ ID NO: 351), permits a UUUU tetraloop and "upper" stem of 7 base pairs but has neither a lower stem nor the consensus 7 nucleotide bulge sequence of la. The upper stem differs from those of la and lb in that it has an unpaired A adjacent to the loop closing G and does not have the invariant GA base pair of la and lb. The affinity of LF1618 is a modest 10 nM which suggests that family Ic forms a less successful structure.

Predictions of minimal high affinity sequences for family 1 ligands can be made and serve as a partial test of the postulated secondary structure. Truncates which include only the upper stem and terminal loop, LF1514T1 (SEQ ID NO: 385) or these two elements plus the 7 nucleotide bulge sequence, LF1514T2 (SEQ ID NO: 386), are not expected to bind with high affinity. On the other hand, there is a reasonable, but not rigorous, expectation that ligands truncated at the base of the lower consensus stem, LF1514T4 (SEQ ID NO: 387) and LF1807T4 (SEQ ID NO: 388), will bind with high affinity. In side by side comparisons, the affinities of LF1514T1 and LF1514T2 for LS-Rg were reduced at least 100-fold in comparison to full length LD1514 (SEQ ID NO: 297), while the affinity of LF1514T4 was reduced less than two fold and that of LF1807T4 approximately three-fold. The correspondence between the predicted and observed truncate affinities supports the postulated secondary structure.

Since the ssDNA ligand LD201T1 (SEQ ID NO: 185) and the adhesion blocking anti-human L-selectin antibody DREG56 are known to bind to the lectin domain of L-selectin, competition between radio-labeled LF1807 (SEQ ID NO: 309) and either unlabeled DREG56 or unlabeled LD201T1 can serve to determine if the 2'-F ligands also bind the lectin domain of purified LS-Rg. In these experiments, both DREG56 and LD201T1 gave concentration dependent inhibition of LF1807 binding. Complete inhibition was attained with 300 nM Mab and 1 RM LD201T1.

The competitors' affinities of LS-Rg, calculated from the competition curves, were in good agreement with their known affinities. These results are consistent with the premise that LF1807, NX280 and DREG56 have the same or overlapping binding sites and consequently it is expected that 2'-F ligands will be antagonists of Lselectin mediated adhesion. These results also reaffirm the proposition that the SELEX protocol, with 5 mM elution of bound oligonucleotides, preferentially elutes ligands bound at or near the lectin domain's bound calcium.

WO 96/40703 PCT/US96/09455 62 Example 26 ssDNA Ligands to Human P-Selectin PS-Rg is a chimeric protein in which the lectin, EGF, and the first two CRD domains of human P-selectin are joined to the Fc domain of a human G1 immunoglobulin Nelson et al., 1993, supra). Purified chimera is provided by A.Varki. Soluble P-selectin is purchased from R&D Systems. Unless otherwise indicated, all materials used in the ssDNA SELEX against the Pselectin/IgG, chimera, PS-Rg, are identical to those of Examples 7 and 13.

The SELEX procedure is described in detail in United States Patent 5,270,163. The specific strategies and procedures for evolving high affinity ssDNA antagonists to P-selectin are described in Examples 7 and 13.

Example 27 2'-F RNA Ligands to Human P-Selectin The Experimental procedures outlined in this Example were used to identify 2'-F RNA ligands to human P-selectin as described in Examples 28-34.

Experimental Procedures A) Materials PS-Rg is a chimeric protein in which the extracellular domain of human Pselectin is joined to the Fc domain of a human G2 immunoglobulin (Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg and CD22P-Rg are analogous constructs of Eselectin and CD22P joined to a human G1 immunoglobulin Fc domain (R.M.

Nelson et al., 1993, supra; I. Stamenkovic et al., 1991, Cell 66, 1133-1144) while LS-Rg has L-selectin joined to an IgG2 Fc domain. Purified chimera were provided by A.Varki. Soluble P-selectin was purchased from R&D Systems. Protein A Sepharose 4 Fast Flow beads were purchased from Pharmacia Biotech. Anti-Pselectin monoclonal antibodies: Gl was obtained from Centocor. The F modified CTP and UTP were prepared according to Pieken et. al. (1991, Science 253:314-317). DNA oligonucleotides were synthesized by Operon. All other reagents and chemicals were purchased from commercial sources. Unless otherwise indicated, experiments utilized HSMC buffer (1 mM CaCl2, 1 mM MgC12, 150 mM NaC1, 20.0 mM HEPES, pH 7.4).

B) SELEX The SELEX procedure is described in detail in United States Patent 5,270,163 and elsewhere. The nucleotide sequence of the synthetic DNA template WO 96/40703 PCT/US96/09455 63 for the PS-Rg SELEX was randomized at 50 positions. This variable region was flanked by N8 5' and 3' fixed regions. The transcript 50N8 has the sequence 3' (SEQ ID NO: 390).

All C and U have 2'-F substituted for 2'-OH on the ribose. The primers for the PCR were the following: N8 5' Primer 5' taatacgactcactatagggagacaagaataaacgctcaa 3' (SEQ ID NO: 197) N8 3' Primer 5' gcctgttgtgagcctcctgtcgaa 3' (SEQ ID NO: 198) The fixed regions include primer annealing sites for PCR and cDNA synthesis as well as a consensus T7 promoter to allow in vitro transcription. The initial RNA pool was made by first Klenow extending 1 nmol of synthetic single stranded DNA and then transcribing the resulting double stranded molecules with T7 RNA polymerase. Klenow extension conditions: 3.5 nmols primer 5N8, 1.4 nmols 40N8, IX Klenow Buffer, 0.4 mM each of dATP, dCTP, dGTP and dTTP in a reaction volume of 1 ml.

For subsequent rounds, eluted RNA was the template for AMV reverse transcriptase mediated synthesis of single stranded cDNA. These single-stranded DNA molecules were converted into double-stranded transcription templates by PCR amplification. PCR conditions were 50 mM KC1, 10 mM Tris-C1, pH 8.3, 7.5 mM MgCl2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of Taq DNA polymerase. Transcription reactions contained 0.5 mM DNA template, 200 nM T7 RNA polymerase, 40 mM Tris-HCl (pH 12 mM MgC12, 5 mM DTT, 1 mM spermidine, 4% PEG 8000, 1 mM each of 2'-OH ATP and 2'-OH GTP, 3.3 mM each of 2'-F CTP and 2'-F UTP, and 250 nM a- 3 2 P 2'-OH ATP.

The strategy for partitioning PS-Rg/RNA complexes from unbound RNA is essentially identical to the strategy detailed in Example 7 for ligands to L-selectin (Table 18).

In the initial SELEX rounds, which were performed at 37 the density of immobilized PS-Rg was 20 pmols/t.l of Protein A Sepharose 4 Fast Flow beads. In later rounds, the density of PS-Rg was reduced (Table 18), as needed, to increase the stringency of selection. Beginning with the second round, SELEX was often done at more than one PS-Rg density. At each round, the eluted material from only one PS-Rg density was carried forward.

Before each round, RNA was batch adsorbed to 100 pl of protein A sepharose beads for 1 hour in a 2 ml siliconized column. Unbound RNA and RNA eluted with minimal washing (two volumes) were combined and used for SELEX WO 96/40703 PCT/US96/09455 64 input material. For SELEX, extensively washed, immobilized PS-Rg was batch incubated with pre-adsorbed RNA for 0.5 to 1 hours in a 2 ml siliconized column with frequent mixing. Unbound RNA was removed by extensive batch washing (500 p1 HSMC/wash). Bound RNA was eluted as two fractions; first, bound RNA was eluted by incubating and washing columns with 5 mM EDTA in HSMC without divalent cations; second, the remaining elutable RNA was removed by incubating and/or washing with 50 mM EDTA in HSMC without divalents. The percentage of input RNA that was eluted is recorded in Table 18. In every round, an equal volume of protein A sepharose beads without PS-Rg was treated identically to the SELEX beads to determine background binding. All unadsorbed, wash and eluted fractions were counted in a Beckman LS6500 scintillation counter in order to monitor each round of SELEX.

The eluted fractions were processed for use in the following round (Table 18). After precipitating with 300 mM Sodium Acetate pH 7 in ethanol volumes), the RNA was resuspended in 80 l of H 2 0 and 40 pl were reverse transcribed into cDNA by AMV reverse transcriptase at 48 o C for 30 minutes, in mM Tris-Cl pH 60 mM NaCI, 6 mM Mg(OAc)2, 10 mM DTT, 200 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/il AMV RT. Transcripts of the PCR product were used to initiate the next round of SELEX.

C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to determine the affinity of RNA ligands for PS-Rg and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 p. pore size, Millipore) were placed on a vacuum manifold and washed with 2 ml of HSMC buffer under vacuum. Reaction mixtures, containing 32 P labeled RNA pools and unlabeled PS-Rg, were incubated in HSMC for 10 20 min at 4 °C, room temperature or 37 filtered, and then immediately washed with 4 ml HSMC at the same temperature. The filters were air-dried and counted in a Beckman LS6500 liquid scintillation counter without fluor.

PS-Rg is a dimeric protein that is the expression product of a recombinant gene constructed by fusing the DNA sequence that encodes the extracellular domains of human P-selectin to the DNA that encodes a human IgG1 Fc region. For affinity calculations, one ligand binding site per PS-Rg monomer (two per dimer) were assumed. The monomer concentration is defined as 2 times the PS-Rg dimer WO 96/40703 PCTIUS96/09455 concentration. The equilibrium dissociation constant, Kd, for an RNA pool or specific ligand is calculated as described in Example 7, paragraph C.

D) Cloning and Sequencing Twelfth round PCR products were re-amplified with primers which contain either a BamHI or a HinDUI restriction endonuclease recognition site. Using these restriction sites, the DNA sequences were inserted directionally into the pUC9 vector. These recombinant plasmids were transformed into E. coli strain JM109 (Life Technologies, Gaithersburg, MD). Plasmid DNA was prepared according to the alkaline hydrolysis method (PERFECTprep, Boulder, CO).

Approximately 50 clones were sequenced using the Sequenase protocol (Amersham, Arlington Heights, IL). The resulting ligand sequences are shown in Table 19.

E) Boundary Experiments The minimal high affinity sequence of individual ligands was determined by boundary experiments (Tuerk et. al. 1990, J. Mol. Biol. 213: 749). Individual RNA ligands, 3 2 p-labeled at the 5'-end for the 3' boundary and 3 2 p-labeled at the 3'-end for the 5' boundary, are hydrolyzed in 50 mM Na2CO3 pH 9 for 8 minutes at 950C. The resulting partial hydrolysate contains a population of end-labeled molecules whose hydrolyzed ends correspond to each of the purine positions in the full length molecule. The hydrolysate is incubated with PS-Rg (at concentrations fold above, below and at the measured Kd for the ligand). The RNA concentration is significantly lower than the Kd. The reaction is incubated at room temperature for minutes, filtered, and then immediately washed with 5 ml HSMC at the same temperature. The bound RNA is extracted from the filter and then electrophoresed on an 8% denaturing gel adjacent to hydrolyzed RNA which has not been incubated with PS-Rg. Analysis is as described in Tuerk et. al. 1990, J. Mol. Biol. 213: 749.

F) 2'-O-Methyl Substitution Experiments In order to decrease the susceptibility of the 2'-F pyrimidine RNA ligands to nuclease digestion, post-SELEX modification experiments were performed to identify 2'-OH purines that are replaceable with 2'-OMe purines without loss of affinity as described in Green et. al. (1995, J. Mol. Biol. 247: 60-68). Briefly, seven oligonucleotides were synthesized, each with three mixed positions. A mixed position is defined as a 2'-OH purine nucleotide within the RNA which has been synthesized with 2:1 ratio of 2'-OH:2'-OMe. Since the coupling efficiency of 2'- WO 96/40703 PCT/IJS96/09455 66 OH phosphoramidites is lower than that of 2'-OMes, the resulting RNA has 25-50 2'-OH at each mixed position. 3 2 P end-labeled RNA ligands are then incubated with concentrations of PS-Rg 2-fold above and 2.5-fold below the Kd of the unmodified ligand at room temperature for 30 minutes, filtered, and then immediately washed with 5 ml HSMC at the same temperature. The bound RNA (Selected RNA) is extracted from the filter and then hydrolyzed with 50 mM Na2CO3 pH 9 for 8 minutes at 95 0 C in parallel with RNA which has not been exposed to binding and filtration (Unselected RNA). The Selected RNA is then electrophoresed on a 20% denaturing gel adjacent to Unselected RNA.

To determine the affect on binding affinity of 2'-OMe substitution at a particular position, the ratio of intensities of the Unselected:Selected bands that correspond to the position in question are calculated. The Unselected:Selected ratio when the position is mixed is compared to the mean ratio for that position from experiments in which the position is not mixed. If the Unselected:Selected ratio of the mixed position is significantly greater than that when the position is not mixed, 2'-OMe may increase affinity. Conversely, if the ratio is significantly less, 2'-OMe may decrease affinity. If the ratios are not significantly different, 2'-OMe substitution has no affect.

G) Cell Binding Studies The ability of evolved ligand pools and cloned ligands to bind to P-selectin presented in the context of a cell surface was tested in experiments with human platelet suspensions. Whole blood from normal volunteers was collected in Vacutainer 6457 tubes. Within 5 minutes of collection, 485 .1 of blood was stimulated with 15 pl Bio/Data THROMBINEX for 5 minutes at room temperature.

A 100 pl aliquot of stimulated blood was transferred to 1 ml of BB- (140 mM NaC1, mM HEPES pH 7.35, 5 mM KC1, 0.01% NaN3) at 4 0 C and spun at 735 x g for minutes. This step was repeated and the resulting pellet was re-suspended in 1 ml of BB+ (140 mM NaCI, 20 mM HEPES pH 7.35, 5 mM KC1, 0.01% NaN3, 1 mM CaC12, 1 mM MgC12) at 4 0

C.

To detect antigen expression, 15 .l BB+ containing FITC conjugated anti- CD61 or PE conjugated anti-CD62 antibody (Becton Dickinson) was incubated for 20-30 minutes at 4 0 C with 10 pl. of platelet suspension. This was diluted to 200 pl with 40C BB+ and analyzed on a Becton Dickinson FACSCaliber using 488 nm WO 96/40703 PCTIUS96/09455 67 excitation and FL1 (530 nm emission) or FL2 (580 nm emission) with the machine live gated on platelets. Between 1000 and 5000 events in this gate were recorded.

To detect oligonucleotide ligand binding, 15 pl BB+ containing ligand conjugated to either FITC or biotin was incubated 20-30 minutes at 4 0 C with 10 pl platelet suspension. The FITC-ligand incubations were diluted to 200 pl with BB+ and analyzed on a FACSCaliber flow cytometer. The biotinylated-ligand reactions were incubated with streptavidin-phycoerythrin (SA-PE) (Becton Dickinson) for minutes at 4 0 C, before dilution and analysis. Wash steps with 500 pl BB+ and 700 x g spins have been used without compromising the quality of the results.

The specificity of binding to P-selectin (CD62P) expressed on platelets was tested by competition with the P-selectin specific blocking monoclonal antibody, G Saturability of binding was tested by self-competition with unlabeled RNA.

H) Inhibition of Selectin Binding to sialyl-Lewisx The ability of evolved RNA pools or cloned ligands to inhibit the binding of PS-Rg to sialyl-Lewisx was tested in competitive ELISA assays Foxall et al., 1992, supra). For these assays, the wells of Coming (25801) 96 well microtiter plates were coated with 100 ng of a sialyl-LewisX/BSA conjugate, air dried overnight, washed with 300 pl of PBS(-) and then blocked with 1% BSA in HSMC for 60 min at room temperature. RNA ligands were incubated with PS-Rg in HSMC/1% BSA at room temperature for 15 min. After removal of the blocking solution, 50 l of PS-Rg (10nM) or a PS-Rg (10nM)/RNA ligand mix was added to the coated, blocked wells and incubated at room temperature for 60 minutes. The binding solution was removed, wells were washed with 300 pl of PBS(-) and then probed with HRP conjugated anti-human IgG, at room temperature to quantitate PS- Rg binding. After a 30 minute incubation at room temperature in the dark with OPD peroxidase substrate (Sigma P9187), the extent of PS-Rg binding and percent inhibition was determined from the OD450.

Example 28 2'-F RNA Ligands to Human P-selectin A. SELEX The starting RNA pool for SELEX, randomized 50N8 (SEQ ID NO: 390), contained approximately 1015 molecules (1 nmol RNA). The SELEX protocol is outlined in Table 18. The dissociation constant of randomized RNA to PS-Rg is estimated to be approximately 2.5 gM. An eight-fold difference was observed in the WO 96/40703 PCT/US96/09455 68 RNA elution profiles with 5 mM EDTA from SELEX and background beads for rounds 1 and 2, while the 50 mM elution produced a 30-40 fold excess over background Table 18. For rounds 1 through 3, the 5 mM and 50 mM eluted RNAs were pooled and processed for the next round. Beginning with round 4, only the mM eluate was processed for the following round. To increase the stringency of selection, the density of immobilized PS-Rg was reduced five fold in round 2 and again in round three without greatly reducing the fraction eluted from the column.

The density of immobilized PS-Rg was further reduced 1.6-fold in round 4 and remained at this density until round 8, with further reductions in protein density at later rounds. The affinity of the selected pools rapidly increased and the pools gradually evolved biphasic binding characteristics.

Binding experiments with 12th round RNA revealed that the affinity of the evolving pool for P-selectin was not temperature sensitive. Bulk sequencing of 2nd, 6th, 11th and 12th RNA pools revealed noticeable non-randomness by round twelve. The 6th round RNA bound monophasically at 37 OC with a dissociation constant of approximately 85 nM, while the 1 1th and 12th round RNAs bound biphasically with high affinity Kds of approximately 100 and 20 pM, respectively.

The binding of all tested pools required divalent cations. In the absence of divalent cations, the Kds of the 12th round pools increased to 10 nM. (HSMC, minus Ca++ plus 2 mM EDTA). The 12th round pool showed high specificity for PS-Rg with measured Kd's of 1.2 gM and 4.9 pM for ES-Rg and LS-Rg, respectively.

B. RNA Sequences In Table 19, ligand sequences are shown in standard single letter code (Comish-Bowden, 1985 NAR 13: 3021-3030). Fixed region sequence is shown in lower case letters. By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise. From the twelfth round, 21 of 44 sequenced ligands were unique. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides.

Sequences that were isolated more than once, are indicated by the parenthetical number, following the ligand isolate number. These clones fall into five sequence families and a group of two unrelated sequences (Orphans)(SEQ

ID

NOs: 199-219).

Family 1 is defined by 23 ligands from 13 independent lineages. The consensus sequence is composed of two variably spaced sequences, WO 96/40703 PCT/US96/09455 69 CUCAACGAMC and CGCGAG (Table 19). In 11 of 13 ligands the CUCAA of the consensus is from 5' fixed sequence which consequently minimizes variability and in turn reduces confidence in interpreting the importance of CUCAA or the paired GAG (see Example 27).

Families 2-5 are each represented by multiple isolates of a single sequence which precludes determination of consensus sequences.

D. Affinities The dissociation constants for representative ligands, including all orphans, were determined by nitrocellulose filter binding experiments and are listed in Table 20. These calculations assume two binding sites per chimera. The affinity of random RNA is estimated to be approximately 2.5 pM.

In general, ligands bind monophasically with dissociation constants ranging from 15 pM to 450 pM at 37 oC. Some of the highest affinity ligands bind biphasically. Full length ligands of families 1-4 show no temperature dependence.

The observed affinities substantiate the proposition that it is possible to isolate oligonucleotide ligands with affinities that are several orders of magnitude greater than that of carbohydrate ligands.

Example 29 Specificity of 2'-F RNA Ligands The affinity of P-selectin ligands to ES-Rg, LS-Rg and CD22P-Rg were determined by nitrocellulose partitioning. As indicated in Table 20, the ligands are highly specific for P-selectin. In general, a ligand's affinity for ES-Rg and LS-Rg is at least 10 4 -fold lower than for PS-Rg. Binding above background is not observed for CD22p-Rg at the highest protein concentration tested (660 nM), indicating that ligands do not bind the Fc domain of the chimeric constructs nor do they have affinity for the sialic acid binding site of this unrelated lectin. The specificity of oligonucleotide ligand binding contrasts sharply with the binding of cognate carbohydrates by the selectins and confirms the proposition that SELEX ligands will have greater specificity than carbohydrate ligands.

Example Inhibition of Binding to sialvl-Lewisx Oligonucleotide ligands, eluted by 2-5 mM EDTA, are expected to derive part of their binding energy from contacts with the lectin domain's bound Ca++ and WO 96/40703 PCT/US96/09455 consequently, are expected to compete with sialyl-Lewis x for binding. In competition assays, the selected oligonucleotide ligands competitively inhibit PS-Rg binding to immobilized sialyl-Lewisx with IC50s ranging from 1 to 4 nM (Table Specifically, ligand PF377 (SEQ ID NO: 206) has an IC50 of approximately 2 nM. Complete inhibition is attained at 10 nM ligand. This result is typical of high affinity ligands and is reasonable under the experimental conditions. The IC50s of ligands whose Kds are much lower than the PS-Rg concentration (10 nM) are limited by the protein concentration and are expected to be approximately one half the PS-Rg concentration. The specificity of competition is demonstrated by the inability of round 2 RNA (Kd- 1 pM) to inhibit PS-Rg binding to immobilized sialyl-Lewisx. These data verify that 2'-F RNA ligands are functional antagonists of PS-Rg.

Example 31 Secondary Structure of High Affinity Ligands In favorable instances, comparative analysis of aligned sequences allows deduction of secondary structure and structure-function relationships. If the nucleotides at two positions in a sequence covary according to Watson-Crick base pairing rules, then the nucleotides at these positions are apt to be paired.

Nonconserved sequences, especially those that vary in length are not apt to be directly involved in function, while highly conserved sequences are likely to be directly involved.

Comparative analysis of the family 1 alignment suggests a hairpin motif, the stem of which contains three asymmetrical internal loops (Figure 16). In the figure, consensus positions are specified, with invariant nucleotides in bold type. To the right of the stem is a matrix showing the number of occurrences of particular base pairs for the positions in the stem that are on the same line. The matrix shows that 6 of the stem's 9 base pairs are supported by Watson-Crick covariation. Portions of the two consensus motifs, CUC and GAG, form the terminus of the stem.

Conclusions regarding a direct role of the terminus in binding are tempered by the use of fixed sequence (11 of 13 ligands) which limits variability. The variability of the loop's sequence and length suggests that it is not directly involved in binding.

This conclusion is reenforced by ligand PF422 (SEQ ID NO: 202) which is a circular permutation of the consensus motif. Although the loop that connects the stem's two halves is at the opposite end relative to other ligands, PF422 binds with high (Kd 172 pM; Table 21) affinity.

WO 96/40703 PCT/US96/09455 71 Example 32 Boundary Experiments Boundary experiments were performed on a number of P-selectin ligands as described in Example 27 and the results are shown in Table 21. The results for family 1 ligands are consistent with their proposed secondary structure. The composite boundary species vary in size from 38-90 nucleotides, but are 40-45 nucleotides in family 1. Affinities of these truncated ligands are shown in Table 22.

In general, the truncates lose no more than 10-fold in affinity in comparison to the full length, effectively inhibit the binding of PS-Rg to sialyl-Lewisx and maintain binding specificity for PS-Rg (Table 22). These data validate the boundary method for identifying the minimal high affinity binding element of the RNA ligands.

Example 33 Binding of 2'-F RNA Ligands to Human Platelets Since the P-selectin ligands were isolated against purified protein, their ability to bind P-selectin presented in the context of a cell surface was determined in flow cytometry experiments with activated human platelets. Platelets were gated by side scatter and CD61 expression. CD61 is a constitutively expressed antigen on the surface of both resting and activated platelets. The expression of P-selectin was monitored with anti-CD62P monoclonal antibody (Becton Dickinson). The mean fluorescence intensity of activated platelets, stained with biotintylated-PF37 7 s1 (SEQ ID NO: 223)/SA-PE (Example 27, paragraph is 5 times greater than that of similarly stained resting platelets. In titration experiments, half maximal fluorescence occurs at approximately 50 pM PF377s1 (EC50) which is consistent with its equilibrium dissociation constant, 60 pM, for PS-Rg. Binding to platelets is specific by the criterion that it is saturable. Saturability has been demonstrated not only by titration but also by competition with unlabeled PF377sl.

Binding to platelets is P-selectin specific by the criteria that 1) oligonucleotides that do not bind PS-Rg do not bind platelets; 2) that binding of PF377sl to platelets is divalent cation dependent; and most importantly 3) that binding is inhibited by the anti-P-selectin adhesion blocking monoclonal antibody G1, but not by an isotype control antibody. These data validate the feasibility of using immobilized, purified protein to isolate highly specific ligands against a cell surface P-selectin.

WO 96/40703 PCT/US96/09455 72 Example 34 2'-O-Methyl Substitution Experiments 2'-OMe purine substitutions were performed on ligand PF377s1 (SEQ ID NO: 223) as described in Example 27 paragraph F and the results are shown in Table 23 The data indicate that 2'-OMe purines at positions 7-9, 15, 27, 28 and 31 enhance binding while substitutions at positions 13, 14, 16, 18, 21 22, 24, and have little or no affect on affinity. Thus it appears that up to 15 positions may be substituted with only slight losses in affinity. In partial confirmation of this expectation, the affinity of 377s 1 simultaneously substituted with 2'-OMe purines at 11 positions (PF377M6, SEQ ID NO: 235) is 250 pM (Table 22).

Example 2'-NH? RNA Ligands to Human P-Selectin The experimental procedures described in this Example are used in Examples 36-38 to isolate and characterize 2'-NH2 RNA ligands to human P-selectin.

Experimental Procedures A) Materials Unless otherwise indicated, all materials used in the 2'-NH2 RNA SELEX against the P-selectin/IgGi chimera, PS-Rg, were identical to those of Example 27.

The 2'-NH2 modified CTP and UTP were prepared according to Pieken et. al.

(1991, Science 253:314-317). The buffer for SELEX experiments was 1 mM CaCl2, 1 mM MgC12, 150 mM NaC1, 10.0 mM HEPES, pH 7.4.

B) SELEX The SELEX procedure is described in detail in US patent 5,270,163 and elsewhere. The nucleotide sequence of the synthetic DNA template for the PS-Rg SELEX was randomized at 50 positions. This variable region was flanked by N8 and 3' fixed regions. The transcript 50N8 has the sequence 5' gggagacaagaauaaac 3' (SEQ ID NO: 248). All C and U have 2'-NH2 substituted for 2'-OH on the ribose. The primers for the PCR were the following: N8 5' Primer 5' taatacgactcactatagggagacaagaataaacgctcaa 3' (SEQ ID NO: 249) N8 3' Primer 5' gcctgttgtgagcctcctgtcgaa 3' (SEQ ID NO: 250). The procedures used to isolate 2'-NH2 oligonucleotide ligands to P-selectin are identical to those described 2'-F ligands in Example 27, except that transcription reactions WO 96/40703 PCTIUS96/09455 73 utilized 1 mM each, 2'-NH2-CTP and 2'-NH2-UTP, in place of 3.3 mM each 2'-F- CTP and 2'-F-UTP.

C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications and in Example 27, paragraph C, a nitrocellulose filter partitioning method was used to determine the affinity of RNA ligands for PS-Rg and for other proteins. Either a Gibco BRL 96 well manifold, as described in Example 23 or a 12 well Millipore manifold (Example 7C) was used for these experiments. Binding data were analyzed as described in Example 7, paragraph C.

D) Cloning and Sequencing Twelfth round PCR products were re-amplified with primers which contain either a BamHI or a HinDm restriction endonuclease recognition site.

Approximately 75 ligands were cloned and sequenced using the procedures described in Example 7, paragraph D. The resulting sequences are shown in Table E) Cell Binding Studies The ability of evolved ligand pools to bind to P-selectin presented in the context of a cell surface was tested in flow cytometry experiments with human platelet suspensions as described in Example 7, paragraph E.

Example 36 2'-NH? RNA Ligands to Human P-Selectin A. SELEX The starting 2'-NH2 RNA pool for SELEX, randomized 50N8 (SEQ ID NO: 248), contained approximately 1015 molecules (1 nmol 2'-NH2 RNA). The dissociation constant of randomized RNA to PS-Rg is estimated to be approximately 6.4 pM. The SELEX protocol is outlined in Table 24.

The initial round of SELEX was performed at 37 °C with an PS-Rg density of 20 pmol/tl of protein A sepharose beads. Subsequent rounds were all at 37 0

C.

In the first round there was no signal above background for the 5 mM EDTA elution, whereas the 50 mM EDTA elution had a signal 7 fold above background, consequently, the two elutions were combined and processed for the next round.

This scheme was continued through round 6. Starting with round seven only the WO 96/40703 PCT/US96/09455 74 mM eluate was processed for the next round. To increase the stringency of selection, the density of immobilized PS-Rg was reduced ten fold in round 6 with further reductions in protein density at later rounds. Under these conditions a rapid increase in the affinity of the selected pools was observed.

Binding experiments with 12th round RNA revealed that the affinity of the evolving pool for P-selectin was temperature sensitive despite performing the selection at 370C, (Kds: 13 pM, 91 pM and 390 pM at 4 room temperature and 37 respectively). Bulk sequencing of RNA pools indicated dramatic nonrandomness at round 10 with not many visible changes in round 12. Ligands were cloned and sequenced from round 12.

B. 2'-NH2 RNA Sequences In Table 25, the 2'-NH2 RNA ligand sequences are shown in standard single letter code (Corish-Bowden, 1985 NAR 13: 3021-3030)(SEQ ID NOS: 251-290). The evolved random region is shown in upper case letters in Table Any portion of the fixed region is shown in lower case letters. By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated otherwise. From the twelfth round, 40/61 sequenced ligands were unique. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides. Sequences that were isolated more than once are indicated by the parenthetical number, following the ligand isolate number.

Ligands from family 1 dominate the final pool containing 16/61 sequences, which are derived from multiple lineages. Families 2 and 3 are represented by slight mutational variations of a single sequence. Sequences labeled as "others" do not have any obvious similarities. Family 1 is characterized by the consensus sequence GGGAAGAAGAC (SEQ ID NO: 291).

C. Affinities The dissociation constants of representative ligands are shown in Table 26.

These calculations assume two RNA ligand binding sites per chimera. The affinity of random 2'-NH2 RNA is estimated to be approximately 10 pM.

At 370C, the dissociation constants range from 60 pM to 50 nM which is at least a x10 3 to 1x10 5 fold improvement over randomized 2'-NH2 RNA (Table 26). There is a marked temperature sensitivity for Clone PA350 (SEQ ID NO: 252) with an increase in affinity of 6 fold at 4 0 C (Table 26). The observed affinities of the evolved 2'-NH2 ligand pools reaffirm our proposition that it is possible to WO 96/40703 PCT[US96/09455 isolate oligonucleotide ligands with affinities that are several orders of magnitude greater than that of carbohydrate ligands.

Example 37 Specificity of 2'-NH, RNA Ligands to P-Selectin The affinity of clone PA350 (SEQ ID NO: 252) for LS-Rg and ES-Rg was determined by nitrocellulose partitioning and the results shown in Table 26. The ligands are highly specific for P-selectin. The affinity for ES-Rg is about 600-fold lower and that for LS-Rg is about 5xl0 5 -fold less than for PS-Rg. Binding above background is not observed for CD22J3-Rg indicating that ligands neither bind the Fc domain of the chimeric constructs nor have affinity for unrelated sialic acid binding sites.

The specificity of oligonucleotide ligand binding contrasts sharply with the binding of cognate carbohydrates by the selectins and reconfirms the proposition that SELEX ligands will have greater specificity than carbohydrate ligands.

Example 38 Cell Binding Studies FITC-labeled ligand PA350 (FITC-350) (SEQ ID NO: 252) was tested for its ability to bind to P-selectin presented in the context of a platelet cell surface by flow cytometry experiments as described in Example 23, paragraph G.

The specificity of FITC-PA350 for binding to P-selectin was tested by competition experiments in which FITC-PA350 and unlabeled blocking monoclonal antibody Gl were simultaneously added to stimulated platelets. Gl effectively competes with FITC-PA350 for binding to platelets, while an isotype matched control has little or no effect which demonstrates that FITC-PA350 specifically binds to P-selectin. The specificity of binding is further verified by the observation that oligonucleotide binding is saturable; binding of 10 nM FITC-PA350 is inhibited by 200 nM unlabeled PA350. In addition, the binding of FITC-PA350 is dependent on divalent cations; at 10 nM FITC-PA350 activated platelets are not stained in excess of autofluorescence in the presence of 5 mM EDTA.

These data validate the feasibility of using immobilized, purified protein to isolate ligands against a cell surface protein and the binding specificity of 2'-NH2 ligands to P-selectin in the context of a cell surface.

Example 39 Inhibition of P-selectin Binding to Sialvl Lewisx In competition assays, ligands PA341 (SEQ ID NO: 251) and PA350 (SEQ ID NO: 252) competitively inhibit PS-Rg binding to immobilized sialyl-Lewisx with ranging from 2 to 5 nM (Table 26). This result is typical of high affinity ligands and is reasonable under the experimental conditions. The IC50s of ligands whose Kds are much lower than the PS-Rg concentration (10 nM) are limited by the protein concentration and are expected to be approximately one half the PS-Rg concentration. The specificity of competition is demonstrated by the inability of round 2 RNA (Kd- 1 pM) to inhibit PS-Rg binding to immobilized sialyl-Lewisx.

These data verify that 2-NH2 RNA ligands are functional antagonists of P-selectin.

Example 2'-NH? RNA Ligands to Human E-Selectin ES-Rg is a chimeric protein in which the extracellular domain of human E- 20 selectin is joined to the Fc domain of a human G1 immunoglobulin Nelson et 1993, supra). Purified chimera were provided by A.Varki. Unless otherwise indicated, all materials used in this SELEX are similar to those of Examples 7 and 13.

The SELEX procedure is described in detail in US patent 5,270,163 and 25 elsewhere. The rationale and experimental procedures are the same as those described in Examples 7 and 13.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Table 1 Wheat Germ Agglutinin Selex Round Total Protein (pmole) Total RNA (PMole) Gel Volume (0i) Total Volume (0i) RNA Eluted RNA Amplified (rim) 5,800 5,800 5,800 5,800 5,800 5,800 580 580 580 58 58 58 2, 020 1,070 1,770 900 500 1,000 1,000 940 192 170 184 180 50 50 50 50 50 50 5 5 5 0.5 0.5 0.5 276 276 280 263 271 282 237 245 265 215 210 210 0.05 0 .12 0.21 3 28.5 28.8 5.7 12 .8 21.4 3.8 5.2 2.3 0 .05 0 .12 0.21 3 28.5 0.18 0 .87 0.64 0.06 0.12 0 .07 6,000,000 600 400 320 260 130 94 68 Table I (Page 2) 0 01 Wheat Germ Lectin Sepharose 6MB, WGA density,approximately 5 mg/ml of gel or 116 PM.

RNA Loading Conditions: Rounds 1-5, 2hrs room temperature on roller; incubation time reduced to 1 hr. for Rounds 6-11.

RNA Elution Conditions: Rounds 1-5, 200 1l of 2 mM (GlcNAc)3, min. room temperature on roller; 2x 200 y1 wash with same buffer.

Rounds 6: 200 ul of 0.2 mM (GlcNAc)3, incubated as above; washed sequentially with 200 1l of 0.5, 1, 1.5, 2 and 10 mM (GlcNAc)3.

Rounds 7-8: 200 il of 0.2 mM (GlcNAc)3, incubated as in round 6; wash twice with same buffer; washed sequentially with 3x 200 p1 each, of 0.5, 1.0, 1.5, 2.0 and 10 mM (GlcNAc)3.

Rounds 9-11: incubated 15 room temperature in 200 p1 of 1 mM (GlcNAc); washed 2x with 200 pl of same buffer; incubation and washes repeated with 2.0 and 10 mM (GlcNAc).

RNA Eluted: percentage of input RNA eluted with (GlcNAc)3 RNA Amplified: percentage of input RNA amplified; Rounds 1-5: entire eluted RNA sample amplified.

Rounds 6-11: pooled 2mM and 10 mM RNA, amplified for subsequent round.

Rounds 9-11: 1.5 mM RNA amplified separately.

0

U

TABLE 2 Wheat Germ Agglutinin 2'NH 2 RNA Ligands Ligand SEQ ID NO. SEQUENCE FAMILY I 11.8 11.4(3) 11.10 11.35 11.5 11.26 11.19 11.15 11.34 6.8 6.9 6.23 6.26 6.14

AUGGUUGGCCUGGGCGCAGGCUUCGAAGACUCGGCGGGAA

CAGOCACUG AAAACUCGGCGGGAA

AGUCUGGCCAAAGACUCGGCGGGAA

GUAGGAGGUUCCAUCACC AGGACUCGGCGGGAA ACAAGGAUCGAUGGCGAGCCOGGGAGG GCUCGGCGGGAA

UUGGGCAGGCAGAGCGAGACCGGGGGCUCGGCGGGAA

AAGGGAUGGGAUUGGGACGAGCGGCC AAGACUCGGCGGGAA aaucauacac aagaCUCGGCGGGAA aaucauacac aagaCUCGGCGGGAA AAGGGAUGGGAUUGGGACGAGCGGCC AAGACUCGGCGGGAA aaucauacaca agaCUCGGCGGGAA CGGGAAUGgcuccgcc CG AAAG UAGUGCCGACUCAGACGCGU

CGUAAAACGGCCAGAAUU

CG GAA, GGUGAUGS CG AAA UCUgcuccgcc CG GAACAGGAAUcgcuccgcc CG AAG CG AAA

CGUGAA

CG AAG CG AAG GGUcgcuccgcc GUGUCAUGGUAGCAAGUCCAAUGGUGGACUCUc

GUGGGUAGGUAGCUGAAGACGGUCUGGGCGCCA

GGUCCgcuccgcc

UGUGUGAGUAACGAUCACUUGGUACUAAAAGCCC

GUGUACUGAAUUAGAACGGUGGGCCUGCUCAUCGU

UGUGGAUGAUAGCACGAUGGCAGYAGUAGUCGGACCGC

GUGAAAGCGUGGGGGGYGCGGGAGGUCUACCCUGAC

aaucauacac aagaCUCGGCGGGAAUCG AAA aaucauacaca agaCUCGGCGGGAAUCGJAA aaucauacacaagaCAGCGGCGG AGUC A

CONSENSUS:

AAGACUCGGCGGGAA CG ?AA FAMILY 2 11.12 11.24(2) 11.27 (2) 11.32 11.6 CGGCUGUGUGUGGU AGCGUCAUAGUAGGAGUCGUCACGAACCAA GGCgcuccgcc CGGCUGU GUGGUGUUGGAGCGUCAUAGUAGGAGUCGUCACGAACCAA GGCgcuccgcc CGAUGCGAGGCAAGAA AUGGAGUCGUUACGAACCC UCUUGCAGUGCGCGc CGUGCGGAGCAAAUAGGGGAUC AUGGAGUCGU ACGAACCGUUAUCGCcgcuccgcc CUGGGGAGCAGGAUAUGAGAUGUGCGGGGCA AUGGAGUCGUGACGAACC gcuccgcc CONSENSUS: CONSESUS:GGAGUCGUGACGAACC TABLE 2 (Page 2) Ligand SEQ ID NO. SEQUENCE0 FAMILY 3 11.13 23 GUCCGCCCCCAGGGAUGCAACGGGGUGGCUCUAAAAGGCUUGGCUAA 11.23 24 GAGAAUGAGCAUGGCCGGGGCAGGAAGUGGGUGGCAACGGAGGCCA 6.3 25 GAUACAGCGCGGGUCUAAAGACCUUGCCCCUAGG AUGCAACGGGGUGCGUCCGCC 6.7 26 UGAAGGGUGGUAAGAGAGAGUCUGAGCUCGUC.CUAGGGAUGCAACGGCACGUCCGCC 6.20 27 CAAACCUGCAGUCGCGCGGUGAAACCUAGGGUUGCAACGGUACAUCGCUGUCGUCCGCC 6.34 28 GUGGACUGGAAUCUUCGAGGACAGGAACGUUCCUAGGGAUGCAACGGACCGUCCGCC 6.35 29 GUGUACCAAUGGAGGCAAUGCUGCGGGAAUGGAGGCCUAGGGAUGCAAC 30 GUCCCUAGGGAUGCAACGGGCAGCAUUCGCAUAGGAGUAAUCGGAGGUC 6.16 31 GCCUAGGGAUGCAACGGCGAAUGGAUAGCGAUGUCGUGGACAGCCAGGU 6.19 32 AUCGAACCUAGGGAUGCAACGGUGAAGGUUGUGAGGAUUCOCCAUUAGGC 6.21 33 GCUAGGGAUGCCGCAGAAUGGUCGCGGAUGUAAUAGGUGAAGAUUGUUGC 6.25 34 GGACCUAGGGAUGCAACGGUCCGACCUUGAUGCGCGGGUGUCCAAGCUAC 6.33 35 AAGGGAGGAGCUAGAGAGGGAAAGGUUACUACGCGCCAGAAUAGGAUGU CONSENSUS: 58 CCUAGGGAUGCAACGG 0 FAMILY 4 11.2 36 CCAACGUA CALJCGCGAGCUGGUG GAGAGUUCAUGA GGGUGUUACGGGGJ 11.33 37 CCCAACGUGUCAUCGCGAGCUGGCG GAGAGUUCAUGA GGGU UACGGGU 11.28 38 GUUGGUGCGAGCUGGGGCGGCGA GAAGGUAGGCGGUCCGAGUGUU CGAAU 11.7(4) 39 aCUGGCAAGRAGUGCGUGAGGGUACGUUAG GGGUGUU UGGGCCGAUCGCAU CONSENSUS: 59 RCUGG GAGRGU GGGUGUU FAMILY 11.20 40 UUGGUCGUACUGGACAGAGCCGUGGUAGAGGGAUUGGGACAAAGUGUCA FAMILY 6 6 .15 41 UGUGAGAAAGUGGCCAACUUUAGGACGUCGGUGGACUGYGCGGGUAGGCUC 6.28 42 CAGGCAGAUGUGUCUGAGUUCGUCGGAGUA GACGUCGGUGGAC GCGGAAC CONSENSUS: Du Ligand SEQ ID NO.

TABLE 2 (Page 3)

SEQUENCE

FAMILY 7 UGUGAUUAGGCAGUUGCAGCCGCC GU GCGGAGACGU GA CUCGAG GAUUC UGCCGGUGGAAAGGCGGGUAGGU GA CCCGAG GAUUCCUACCAAGCCAU GAGGUGRA UGGGAGAGUGGAGCCCGGGUGACUCGAGGAUUCCCGU 6.24 6.27 11.3

CONSENSUS:

GGGNNNGU GA CYCGRG GAYUC FAMILY 8 6.2 6.6(2) GUCAUGCUGUGGCUGAACAUACUGGUGAAAGUtJCAGUAGGGUGGAUACAgcuccgCcc CCGGGGAUGGUGAGUCGGGCAGUGUGACCGAACUGGUGCCCGCUGAGAgcuc c

CONSENSUS:

UGANCNNACUGGUGNNNGNGNAG

FAMILY 9 6.11 6.13 6 .17 ACACUAACCAGGUCUCU GAACGCGGGAC GGAGGUG UGGGCGAGGUGGAA CCGUCUCCCGAGAACCAGGCAGAGGACGUGCUGAAGGAGCUG CAUCUAGAA CCGUCUCC GAGAACCAGGCAGAGGAGGUGCUGAAGGRGCUGGCAUCUACAA CONSENSUS: GUCUCY GAACNNGGNA GGANGUGNUG GAGNUG

ORPHANS

6.4 6.18 11 .30 11 .29

CCCGCACAUAAUGUAGGGAACAAUGUUAUGGCGGAAUUGAUAACCGGU

CGAUGUUAGCGCCUCCGGGAGAGGUUAGGGUCGUGCGGNAAGAGUGAGGU

GGUACGGGCGAGACGAGAUGGACUUAUAGGUCGAUGAACGGGUAGCAGCUC

CGGUUGCUGAACAGAACGUGAGUCUUGGUGAGUCGCACAGAUUGUCCU

ACUGAGUAAGGUCUGGCGUGGCAUUAGGUUAGUGGGAGGCUUGGAGUAGc t0

(A

WO 96/40703 WO 9640703PCTIUS96/09455 82 Table 3 Dissociation Constants of RNA Ligands to WGA Ligand SEQ ID NO: Kd Family 1 11.8 4 9.2 nM 11.4 5 32 nM 11.35 7 11.5 8 44 nM 11.26 9 38 nM 11.19 10 22 nM 11.15 11 54 nM 11.34 12 92 nM 6.8 13 11mM 6.9 14 396 nM 6.23 15 824 nM 6.14 17 Family 2 11.12 18 15.2 nM 11.24 19 19.4 nM 11.27 20 30 nM 11.32 21 274 nM 11.6 22 702 nM Family 3 11.13 23 11.23 24 6.3 25 120 nM 6.2 27 6.34 28 6.35 29 30 678 nM 6.16 31 6.19 32 74 nM Family 4 11.2 36 62 nM 11.33 37 11.28 38 9.2 nM 11.7 39 16 nM WO 96/40703 WO 9640703PCTIUS96/09455 83 TABLE 3 (Page 2) Ligand SEQ ID NO: Family 11.2 1.4 nM Family 7 6.27 11.3 56 nM 410 nM Family 8 Family 9 6.11 Orphans 11.3 11 .29 56 nM 32 nM The Kds of ligands that show 5 binding at 1 jiM WGA is estimated to be 20 jiM.

WO 96/40703 PTU9195 PCTfUS96/09455 84 Table 4 Specificity of RNA Ligands to WGA Kds for N-acetyl-glucosamine Binding Lectins

LECTIN

Triticum vulgare (WGA) Canavalia ensiformis (Con Datura stramonium Ulex europaeus (UEA-II) Ligand 6.8 (SEQ ID NO:13) 11.4 nM 4.4 jiM Ligand 11.20 Ligand 11.24 (SEQ ID NO:40) (SEQ ID NO:19) 1.4 nM 19.2 nM 11.2 jiM 2.2 jiM *Less than 5% binding at 1 jiM protein; estimated Kd 20 jiM *succinylated Con A_ WO 96/40703 WO 9640703PCT/US96/09455 TABLE INHIBITION OF R~NA LIGAND BINDING TO WIFAT GERM AGGULTININ Lia~n SEO ID NO: cometitor ICo..A4Mf Max Inhib 6.8 11.20 11.24 13 (GlcNAc) 3 40 (GlcNAc) 3 19 (GlcNAc) 3 95 120 120 95 95 95 10.9 8.4 19.4 Kc is the dissociation constant of (GlcNAc)3 calculated from these data, assuming competitive inhibition and two RNA ligand binding sites per dimer.

WO 96/40703 WO 9640703PCT/US96/09455 86 Table 6 INHIBITION OF WGA MEDIATED AGGLUTINATION OF SHEEP ERYTHROCYTES inhibitor 6.8 11.20 11.24 (GlcNAc) 3 GlcNAc Inhibitory Concentration (pM) BEO ID NO: Comvlete Partial 13 0.5 0.12 40 0.5 0.12 19 *2 8 2 780 200 Complete inhibition of agglutination by ligand 11.24 was not observed in this exomeriment.

TABLE 7a 1-Selectin 2'NH9-RNA SELEX at 4 OC SELEX Total Round RNA pmoles Rnd 0 Rnd 1 Rnd 2 Rnd 3 Rnd 4 Rnd 5 Rnd 6 Rnd 7 Rnd 8 Rnd 9 Rnd 10 Rnd 11 Rnd 12 Rnd 13 Rnd 14 1060 962 509 407 429 439 452 46 43 44 22 43 23 23 21 23 30 12 7 9 Total Protein pmoles 167.0 167.0 1 67.0 167.0 1 67.0 16.7 167.0 16.7 16.7 16.7 4.2 16.7 4.2 4.2 4.2 8.4 10.5 10.5

RNA:LS-

Rg Ratio 6.3 5.8 3.0 2.4 2.6 26.3 2.7 2.8 2.6 2.6 5.2 2.6 5.5 5.5 5.0 2.7 2.9 1 .1 7 9 Bead Volume I OgL 1 OjiL 1 0OiL 1OfiL 1 OjiL 1 0OiL IlOpL 1 0~tL IlOpiL 250[tL 250piL 250pL 250piL Total -l1OOpiL -lOOpiL -lOOpiL -loopiL -lO I 0L -lOOpiL -100ptL -IOOiiL -lO0pL 1 OOpiL 10011 OOpiL -lIOOpIL -lOOpiL -iS5piL -500tLL -50iL -500gL -500p.L 5mM

EDTA

Eluted

RNA

0.498 0.306 1.480 5.010 8.357 0.984 7.409 3.468 8.679 7.539 2.748 8.139 2.754 4.352 6.820 14.756 0.707 3.283 4.188 4.817 50mM

EDTA

Eluted

RNA

0.301 0.114 0.713 1.596 7.047 0.492 6.579 1.312 2.430 2.358 1 .298 1.393 0.516 0.761 1.123 1 .934 0.033 0.137 0.136 0.438 Kd (nM) 10,000 434 133 37 33 13 0.3 0.7 0O TABLE 7a (Page 2) L-Selectin Rg was immobilized on Protein A Sepharose 4 Fast Flow. Protein A density is approximately 6mg/ml drained gel (143iM).

RNA Loading Conditions: All selections were carried out in the cold room. The RNA used in each selection was first incubated for minutes with 100RL Protein A Sepharose in the cold room on a roller. Only RNA which flowed through this column was used on the LS-Rg selection column. The RNA was incubated on the selection column for minutes on a roller before being washed extensively with binding buffer (20mM HEPES pH7.4 150mM NaCI, 1mM MgCI2, 1mM CaCl2.) RNA Elution Conditions: RNA was eluted by incubating the extensively-washed columns in 100L of HEPES buffered EDTA (pH7.4) for 30 minutes on a roller followed by three 100pL HEPES buffered EDTA washes.

'C

TABLE 7b Temperature L-Selectin 2'NH9-RNA SELEX at Room

SELEX

Round Rnd 7 Rnd 8 Rnd 9 Rnd 10 And 11 Rnd 12 Rnd 13 Total

RNA

pmoles 43 35 24 30 1 2 6 1 Total Protein pmoles 10.0 10 10 2.5 6.3 6.3 0.6 0.6

RNA:LS-

Rg Ratio 4.3 3.5 3.5 9.6 4.9 1.9 9.4 1.4 Bead Volume 10OgL lOjtL 1l0jtL I 0gL 250ptL 25OpL 25OilL 250piL Total 5M Volume ETA

RNA

1 lOOiiL 1.205 I 100jiL 6.642 I-lOOjiL 5.540 -100gL 1.473 -500iiL 0.707 -500[LL 3.283 -500glL 0.877 -500[tL 5.496 50mM

EDTA

Eluted

RNA

0.463 0.40 1 0.39 1 0.383 0.033 0.134 0.109 0.739 Kd (nM) 13 0.3 0.7 L-Selectin Rg was immobilized on Protein A Sepharose 4 6mg/mI drained gel (1 43p M).

Fast Flow. Protein A density is approximately RNA Loading Conditions: Selections were carried out at room temperature. The RNA used in each selection was first incubated for minutes with 100ptL Protein A Sepharose at room temp. Only RNA which flowed through this column was used on the LS-Rg selection column. The RNA was incubated on the selection column for 90 minutes on a roller before being washed extensively with binding buffer (20mM HEPES pH7.4 150mM NaCI, 1mM MgCI2, 1mM CaCl2.) RNA Elution Conditions: RNA was eluted by incubating the extensively-washed columns in lO0jiL of HEPES buffered EDTA (pH7.4) for 30 minutes on a roller followed by three l00[tL HEPES buffered EDTA washes.

TABLE 8 L-Selectin 2'NH2 RNA LIGANDS Ligand SEQ ID NO.

Sequences Family I F13 .32 (5) 6.60(2) 6.50 (3) 6.79 F14. .9 F14 .21 F14 .25 F13 48 (2) 6.71 6.28

CONSENSUS:

67 68 69 70 71 72 73 74 75 76 118 CGCGUAUGUGUGAAAGCGUGUGCACGGAGGCGU- CUACAAU

GGCAUUGUGUGAAUAGCUGAUCCCACAGGUAACAACAGCA

UAAUGUGUGAAUCAAGCAGUCUGAAUAGAUUAGACAAAAU

AUGUGUGAGUAGCUGAGCGCCCGAGUAUGAWACCUGACUA

AAACCUUGAUGUGUGAUAGAGCAUCCCCCAGGCGACGUAC

UUGAGAUGUGUGAGUACAAGCUCAAAAUCCCGUUGGAGG

UAGAGGUAGUAUGUGUGGGAGAUGAAAAUACUGUGGAAAG

AAAGUUAUGAGUCCGUAUAUCAAGGUCGACAUGUGUGAAU

CACGAAAAACCCGAAUUGGGUCGCCCAUAAGGAUGUGUGA

GUAAAGAGAUCCUAAUGGCUCGCUAGAUGUGAUGUGAAAC

AUGUGUGA

Family 11 F14. .20 (26) F 14. 12 (2 2) F14 .11(12) F13 .45 (9) 6.80

CONSENSUS:

UAACAA CAAUCAAGGCGGGUUCACCGCCCCAGUAUGAGUG UAACAA CAAUCAAGGCGGGUUYACCGCCCCAGUAUGAGUA UAACAA CAAUCAAGGCGGGUUYACCGCUCCAGUAUGAGUA UAACAA CAAUCAAGGCGGGUUCACCGCCCCAGUAUGAGUG ACCAAGCAAUCUAU GGUCGAACGCUACA CAUGAAUGACGUc CAA CAAUC AUGAGUR TABLE 8 (Page 2) Ligand SEQ ID NO.

6 .17 6.49 6.16 6.37 6.78 6.26 6.40 F13 57

CONSENSUS:

SEQUENCE

Family III GAACAUGAAGUAAUCAAAGUCGUACC

AAUAUACAGGAAGC

GAACAUGAAGUAAGAC CGUCAC AAUUCGAAUGAUUGAAUA GAAC UGAAGUAAAA AGUCGACG AAUUAGCUGUAACCAAAA GAACAUGAAGUAAA AGUCUG AGUUAGUAAAUUACAGUGAU GAACUUGAAGUUGA ANUCGCUAA GGUUAUGGAUUCAAGAUU AACAUGAAGUAAUA AGUC GACGUAAUUAGCUGUAACUAAA AACAUGAAGUAAA AGUCUG AGUUAGAAAUUACAAGUGAU- UAACAUAAAGUAGCG CGUCUGUGAGAGGAAGUGCCUGGAU AACAUGAAGUA AGUC ARUUAG Family IV 6.58 6.69

CONSENSUS:

90 91 121

AUAGAACCGCAAGGAUAACCUCGACCGUGGUCAACUGAGA

UAAGAACCGCUAGCGCACGAUCAAACAAAGAGAAACAAA-

AGAACCGC WAG Family V 6.56 F13 .55

CONSENSUS:

F14 .27 F14 .53

CONSENSUS:

UUCUCUCCAAGAACYGAGCGAAUAAACSACCGGASUCACA

UGUCUCUCCUGACUUUUAUUCUUAGUUCGAGCUGUCCUGG

UcUcUCC Family VI CCGUACAUGGUAARCCU CGAAGGAUUCCCGGGAUGAUCCC

UCCCAGAGUCCCGUGAUGCGAAGAAUCCAUUAGUACCAGA

CGAAGAAUYC

Family VII F13 .42 F13 .51

CONSENSUS:

GAUGUAAAUGACAAAUGAACCUCGAAAGAUUGCACACUC

AUGUAAAUCUAGGCAGAAACGUAGGGCAUCCACCGCAACGA

AUGUAAAU

TABLE 8 (Page 3) Ligand SEQ ID NO. SEQUENCE Family VIII 6.33 (11) 6.41

CONSENSUS:

98B 99 125

AUAACCCAAGCAGCNIJCGAGAAAGAGCUCCAUAGAUGAU-

CAAAGCACGCGUAUGGCAUGAAACUGGCANCCCAAGUAAG

AACCCAAG

Family IX F13. .46 (4) 100

CAAAAGGUUGACGUAGCGAAGCUCUCAAAAUGGUCAUGAC

Family X F14 2 F14.13 (2) 101 102 AAGUGAAGCUAAAGCGGAGGG CCAUUCAGUUUCNCACCA AAGUGAAGCUAAAGSGGAGGG CCACUCAGAAACGCACCA Family XI 6.72 (2) 6 .42

CACCGCUAAGCAGUGGCAUAGCCCAGUAACCUGUAAGAGA

CAC-GCUAAGCAGUGGCAUAGC- -GWAACCUGUAAGAGA Family XII 6.30(5) 6.52 (2) AGAUUACCAUAACCGCGUAGUCGAAGACAUArJAGUAGCGA Family XIII 106 106 ACUCGGGUAGAACGCGACUUGCCACCACUCCCAUAAAGAC 0 TABLE 8 (Page 4) Ligand SEQ ID NO.

SEQUENCE

orphans 6.14 107

UCAGAACUCUGCCGCUGUAGACAAAGAGGAGCUUAGCGAA

6.36 108

AAUGAGCAUCGAGAGAGCGCGAACUCAUCGAGCGUACUA

6.41 119

CAAAGCACGCUAUGGCAUGAACUGGCNCCCAGUAAG

6.44 110

GAUGCAGCAACCUGAAAACGGCGUCCACAGGUAUACAG

6.70 ill

AAACUCGCUACAAACACCCAAUCCUAGAACGUUAUGGAGA

6.76 112

CUAGCAUAGCCACCGGAACAGACAGAUACGAGCACGAUCA

6.99 113

GAUUCGGAGUACUGAAAACAACCCUCAAGUGCAUAGG

6.81 114

GUCCAGGACGGACCGCAGCUGUGAUACAAUCGACUUACAC

6.70 115

AAACUCGCUACAAACACCCAAUCCUAGAACGUUAUGGAGA

F13 .59 116

CGGCCCUUAUCGGAGGUCUGCGCCACUAAUUACAUCCAC

F14 .70 117

UCCAGAGCGUGAAGAUCAACGUCCCGGNGUCGAAGA

WO 96/40703 WO 9640703PCT/US96/09455 94 TABLE 9 Dissociation Constants of 2' NH 2 RNA Ligaxids to L-Selectin* QWnTn NO:l A 0 c Rm Temn

U

Family 3I F13 .32 F13.48 F14.9 F14.21 F14.25 15. 7 nM 15.9 nIM 8. 2 DM 2. 3 nM 1300 nM 14. 9 nM 9. 2 nM 15.4 DIM 15. 9 nM Family 11 F14.12 F14.20 5. 8 PM (0.68) 16.2 DIM 58 pM (0.68) 6 0 rLM 1. 7 niM (0.62) 94 nM 1. 0 IM (0.28) 48 nM Family III 3 .0 nLM F13.57 75 nM Family V F13 .55 62 pM 1.5 r24 F14.53 F14.27 Family VI 97 pM (0.65) 14. 5 riM 14 5 rJM Family VII 2.0 n14 8 .8 ii14 142 1214 F13 .42 F13.51 5. 5 n14 18 rim WO 96/40703 WO 9640703PCT1US96/09455 TABLE 9 (Page 2) 1 3 own Rm TemD ID NO: 4 OC Family X F14.2 F14.13 101 102 1. 8 rim 1. 3 n~M (0.74) 270 tnM 7. 2 nM orphans F13 .59 F14.70 116 117 5% 2 .0 niM (0.75) 254 tiM 7. 8 riM (0.58) 265 nM Kds of monophasic binding ligands are indicated by a single number; the high affinity Kd Kdj), the mole fraction binding with Kdj, and the low affinity Kd Kd2) are presented for biphasic binding ligands.

WO 96/40703 WO 9640703PCTIUS96/09455 96 TABLE Specificity of 2' NH 2 RNA Ligands to L-Selectin* -r 4 rA OWN T% wTA. T.Q-10 Ir Cz 'D -C er rnl> 1> -1D Family I F13 .32 F13 .48 F14.9 F14.21 F14.25 15.7 niM 15. 9 rli 8. 2 n.M 2. 3 n.M 1300 n.m 5% 5% 2. 6 p.M 17 JIM 720 rli Family 11 F14.12 F14.20 60 pM 58 pM (0.68) n.M 47 n.M 7 0 n.M 910 rim Family III F13 .57 3. 0 r.M 2. 7 JM Family V F13.55 62 pM 49 n.M Family VI 5. 8 gM 5. 2 JM F14.53 97 pM (0.65) 14. 5 n.M 355 n.M Family VII F13 .42 F13.51 2 .0 n.M 8. 8 nM 4. 4 pM 2. 0 jM Family X F14.2 101 18nM 19jM 40nM <5 1. 8 nM- 1. 9 PM 450 nM WO 96/40703 WO 9640703PCT/US96/09455 97 Table 10 (Page 2) C'Wn 'M Wn VRg-Rca PS-Rca CD2 2-Rar J. %4 LS-'Rer orphans F13.59 F14.70 116 117 5% 2. 0 nM (0.75) 254 nM 5% 5. 9 PIM Dissociation constants were determined at 4 0 C in HSMC buf fer.

When 5% binding was observed at the highest protein concentration, the Kd is estimated to be 20 pM~.

Table 11 L-SELECTIN ssDNA SELEX Round Temp. Total DNA Total Prot. DNA:Proteln Bead Vol. Total Vol. Eluted 2 mM EDTA Eluted 50 mM EDTA Kd, nM 4 degrees slgnal:bkgd 2 mM Rnd 0 Rnd 1 Rnd 2 Rnd 3 Rnd 4 Rnd 5 Rnd 6 Rnd 7 Rnd 8 Rnd 9 Rnd1O Rnd 11 Rnd 12 Rnd 13 Rnd 14 Rnd 15 pmol 930 400 460 100 1 00 1 000 100 100 10 10 2.5 2.5 2.5 5 1 .25 pmol 1 67 167 167 16.7 16.7 16.7 16.7 16.7 1.67 1.67 0.42 0.42 0.42 0.84 0.21 1OgL lOpL 10 1 iL 1 OjiL lOp~L 50 1 iL -100 [tL -100 jiL -100 p.L -100 VIL -100 VIL 100 p~L -100 VIL -100 lIL -100 gL -100 VIL -100 piL -100 pL -100 piL -100 pL -100 piL n/a n/a n/a 0.35 0.23 1.42 6.9 1 .9 0.5 2.2 0.37 0.86 0.7 2.8 1 .7 5.5 2.19 2.55 0.29 0.08 0.38 0.93 0.31 0.16 0.57 0.07 0.13 0.35 0.76 0.5 10,000 967 60 2.1 1.3 25 0

C

0.44 25 'C 0.16 25 DC 12 1.3 3 4 18 9 1.6 8 11 4 7 Binding Buffer, Rounds 1-9 10 mM HIEPES, pH at room temp w/NaOH to 7.4 100 mM NaCI 1mnM MgCI2 1 mM CaCI2 mnM KOP Elution Buffers: replace divalent cations with EDTA TABLE 12 L-Selectin SSDNA Ligands Family 1 Ligand SEQ ID NO SEQUENCE LD204 (3) LD1 45 LD183 (2) LD2 30 (2) LD208 (7) LD227 (5) LD1 12 LD1 37 LD179 (2) LD182 LD1 90 LD193 (2) LD199 LD201 (2) LD203 LD207 LD216 LD233 (5) LD191 LD128 (3) LD111 (2) LD1 39 LD237 LD173.

LD209 LD2 21 LD108 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155

GGAACACGTGAGGTTTAC

CCCCGAAGAACATTTTAC

GGCATCCCTGAGTC-ATTAC

TGCACACCTGAGGGTTAC

CACGTTTC

CGGACATGAGCGTTAC

CGCATCCACATAGTTC

TACCCCTTGgGCCTCATAGAC

CACATGCCTGACGCGGTAC

TAGTGCTCCACGTATTC

AGCGATGC

CCAGGAGCACAGTAC

ACCACACCTGGqCGGTAC CAAGGTAACCgAGTAC

ACCCCCGACCCGAGTAC

CIAGTAC

ACAACG-AGTAC

CACGACAGAGAAC

AGGGAGAAC

AGGACC

gicTACAC

GGAC

cicTAC!AC

CGGCTATAC

GAGTAGCC

GAGTAGCC

GAGTAGCC

AAGGCACTCGAC

AAGGTGCTAAAC

AAGGTTCTTAAC

AAGGCGCTAGAC

AAGGGGTTACAC

AAGGTGCTAAAC

AAGGGGCTACAC

AAGGTCTTAAAC

AAGGCCTGG AC

AAGGTGCTAAAC

AAGGGGCTACAC

AAGGTGTTAAAC

AAGGAGTTATCC

AAGGTGCTAAAC

AAGGCATTCGAC

AAGGTGTTAAAC

AAGGAGATAGAC

AAGGCGTTAGAC

AAGGTGCTAAAC

AAGGTGTTAAAC

AAGGTGCTAAAC

AAGGCACTCGAC

AAGGGGCCAAAC

NNGGTGCTAAAC

AAGGCGTTAGAC

AAGGCGTTAGAC

AAGGCGTTAGAC

9MAAACACTT

GTA-AAATCAG

GTALTQTAC

9TA&C9TCTC

GALMCATTCACTCCTTGGC

aTAACGTACTT

GAAATATTGCA

GTTAGC

GTAACGTTG

GAAGACGGCCT

GCAACGATTTAGATGCTCT

gTAATGTCTGGT

GTAACGTGT

GTAATGGCTPCG

GTAATCTGGT

aTAATGCCGATCGAGTTGTAT gTAATCGGCGCAGGTATC

GTTATCCGACCACG

GTTTATCTACACTTCACCT

GGCTCCCCTGGCTATGCCTCTT

GTAGAGCCAGATCGGATCTGAGC

GTAGTTTATAACTCCCTCCGGgCC 9GAGACCCAGACGCGGATCTGACA

GCAGAGACTCGATCAACA

GGAGGGGGAATGGAAGCTTG

GGAGGGGGAATGG

GGAGGGGGAATGTGAGCACA

TABLE 12 (Page 2) SEQUENCE Ligand SEQ ID NO LD 141 39 LD547 LD516 LD543 45 156 175 176 177 178 179 Truncates LD2O1T1 LD2O1T3 LD201T4 LD2O1T1O LD201T12 LD227t5 LD227x1 LD227t1 NX288 NX303 Consensus: TAGCTCCACACAC AASSCGCRGCAC CGGCAGGGCACTAAC AAGGTGTTAAAC TGCACACCGGCCCACCCGGAC AAGGCGCTAGAC GACGAAGAGGCC AAGGTGATAACC AAGGACTTAGCTATCC AAGGCACTCGAC ATGCCCAGTTC AAGGTTCTGACC tagcCAAGGTAACCAGTAC AAGGTGCTAAAC GTAACCAGTAC AAGGTGCTAAAC CfAGTAC AAGGTGCTAAAC CGCGGTAACCAGTAC AAGGTGCTAAAC GCGGTAAgAGTAC AAGGTGCTAAAC ACATGAGCGTTAC AAGGTGCTAAAC cgcGCGTTAC AAGGTGCTAAAC GCGTTAC AAGGTGCTAAAC dttagcCAAGGTAACCAGTAC AAGGTGCTAAAC dtC CAGTAC AAGGTGCTAAAC TAC AAGGYGYTAVAC

ATAGGGGATATCTGG

GTTACGGATGCC

GAAATGACTCTGTTCTG

GGAGTTTCCGTCCGC

GAAGAGCCCGA

GAAATGACTCTGTTCTG

GTAATGGCTTCGgct tac OTMTGGCTTCGgcttac

QTATGG

aTAATGGCGCG QTAACGTACTTgct tactctcatgt gTAACGTACTTgct tac tcgcg

GTAACGT

GTAATGGCTTCGgcttact[3'3'It GTAAhTGGt [3'3')t

GTA

Family 2 LD181 157 CAT CAAGGACTTTGCCCGAAACCCTAGGTTCACG TGTGGG Family 4 LD174 (2) LD122 LD239 LD533 Truncates LD1'74t1 158 159 160 180

CATTCACCATGGCCCCTTCCTACGTATGTTCTGCGGGTG

GCAACGTGGCCCCGTT TAGCTCATTTGACCGTTCCATCCG CCACAGACAATCGCAGTCCCCGTG TAGCTCTGGGTGTCT GCAGCGTGGCCCTGTT TAGCTCATTTGACCGTTCCATCCG 194 tagcCATTCACCATGGCCCCTTCCTACGTATGTTCTGCGGGTGgctta Consensus: 182 GCCG GGCCCCGT TABLE 12 (Page 3) Family Ligand SEQ ID NO SEQUENCE LD1 09 161 CCACCGTGATGCACGATACATGAQQGGTGTCAGCGCAT LD1 27 162 CGAGGTAGTCGTTATAGGGTGCGCACGACACACAGCGGTRG Consensus: 183 RCACGAYACA Family 6 LD1 96 163 TGGCGGTACGGGCCGTGCACCCACTTACCTGGGAAGTGA LD229 164 CTCTGCTTACCTCATGTAGTTCcAAGCTTGGCGTAATCATG Truncate LD1 96t 1 195 agcTGGCGGTACGGGCCGTGCACCCACTTACCTGGGAAGTGAgctta Consensus: 184 CTTACCT 0 Family 7 LD206(2) 165 AGCGTTGT ACGGGGTTACAC ACAACGATTTAGATGCTCT orphans LD2 14 166 TGATGCGACTTTAGTCGAACGTTACTGGGGCTCAGAGGACA LD1 02 167 CGAGGATCTGATACTTATTGAACATAMCCGCACNCAGGCTT LD530 168 CGATCGTGTGTCATGCTACCTACGATCTGACTA 04 169 GCACACAAGTCAAGCATGCGACCTTCAACCATCGACCCGA 09 170 ATGCCAGTGCAGGCTTCCATCCATCAGTCTGACANNNNNN 23 171 CACTTCGGCTCTACTCCACCTCGGTCCTCCACTCCACAG- LD527 172 CGCTAACTGACCCTCGATCCCCCCAAGCCATCCTCATCGC 41 173 ATCTGACTAGCTCGGCGAGAGTACCCGCTCATGGCTTCGGCGAATGCCCT LD548 174 TCCTGAGACGTTACAATAGGCTGCGGTACTGCAACGTGGA t0 th WO 96/40703 PTU9/95 PCTIUS96/09455 102 Table 13 Dissociation Constants of ssDNA Ligands to L-Selectin Licrand Family LD.111 LD12 8 LD108 LD112 LD13 7 LD13 9 LD145 LD1 79 LD1 82 LD1 83 LD193 LD2 01 LD2 04 LD2 08 LD2 27 LD230 LD2 33 Family LD181 Family LD122 LD174 LD2 39 Family LD109 LD127 SEO ID NO: 149 148 155 135 136 150 130 137 138 131 140 142 129 155 134 132 146 157 159 158 160 161 162 Room Temiperature 330 pM 310 pM 160 pM 300 pM 520 pM 210 pM 920 pM 180 pM 130 pM 170 pM 88 pM 110 pM 100 pm 110 pM 43 pM 57 pM 110 n.M 84 pM 1. 8 n.m 43 pM 170 pM 190 pM 1. 0 n.M 37 0 C 11. 8 nM 1.8 nM 8.5 nM 23.2 raN 0.65 inM 6.8 nMY 8.8 nM 590 pM 2.0 riM 1.0 rim 970 pM 1.2 riM 3.7 riM 380 pM 160 pM 260 pM 380 pM 1. 8 rM 2. 1 riM 370 pM 1. 6 rM 9. 6 rM 890 pM WO 9640703PCTIUS96/09455 WO 96/40703 103 TABLE 13 (Page 2) Licrand SEO ID NO: Family 6 LD196 Family 7 LD2 0 6 orphans LD1 02 LD2 14 163 165 167 166 Temperature 13 0 pM 330 pM not determined 660 pM 37 0 C 3. 4 rM 6. 0 rM 7. 9 nM 8.4 nM 660 pM 4.8 n.M 43 nM Round 15 Pool LD2QlTl* LD20lT3* 160 PM *LD2O1T and LD2OlT3 were made by solid state of the synthetic full length LD201 control was of enzymaticall~y synthesized LD201 was 1.8 riM.

synthesis; the Kd 3.8 riM while that WO 96/40703 WO 9640703PCT/TJS96/09455 104 Table 14 Specificities of ssDNA Ligands to L-Selectin* Ligand Family LD1 11 LD201 LD204 LD227 LD230 LD233 Family LD 181 Family LD122 LD174 Family LD127 Family LD 196 Family LD206 SEQ ID NO: 149 142 129 134 132 146 157 159 158 LS-Rg ES-Rg 1.1 nM 1.2 j±M llOnM 37 nM 450 pM 1.5 PM 64 pM 33 nM 44 pM 19 nM 120 pM 39 nM 200 pM 37 nM 340 pM 400 nM 46 pM 28 rNM 250 pM 1.3 gM 220 pM 50 nM 120 pM 100 nM 840 nM 1.0 g±M 2.9 gM 560 nM 600 nM 420 nM 1.6 p.M 420 nM 380 nM 780 nM 3.4 p.M 600 nM *Kds were determined at room temperature. In assays with 700 nM CD22 B-Rg and 1.4 p.M WGA less than 1% and 3% binding, respectively, was observed for all ligands suggesting that the dissociation constants are greater than 100 p.M for these proteins.

WO 96/40703 WO 9640703PCT/US96/09455 105 Table Summary of Selection Conditions and Results from 2'F RNA Human L-selectin- SELEXes 30n7 2'Fluro SELEX SELEX Total Round RNA pm ole 5 1 630 2 656 3 608 4 193 193 6 86 7 17 8 17 9 19 18 11 103 11 27 12 89 12 79 Total Protein pm ole s 100 100 100 20 20 10 2 2 2 2 12.5 2.5 5 5 Temp.

Time, Vol.

37 0 C. 15' 10W± 37'C. 15'1041~ 37 0 C. 15' lOPIl 37 0 C. 15' 101±1 37 0 C. 15' 101±1 37 0 C. 15' 10J.L 37 0 C. 15' l~p.1 37 0 C. 15' 101.1 37 0 C. 15' 101±1 37 0 C. 15' 101.1 37 0 C. 15' 50j±1 37 0 C. 15' 501±1 37 0 C. 15' S5jil 37 0 C. 15' S0ul Bound

LS-R

Sites 0.7 2.8 11.6 7.4 19.7 15.7 12.1 55.1 40.1 28.4 647.7 63.1 53.2 54.8 5mM

EDTA

El uted 0.1 0.4 1.9 0.8 2.1 1.9 1.4 6.6 4.2 3.3 8.3 5.9 3.0 3.5

EDTA

Signal/ Bkgnd 24 68 24 17 8 3 2 4 3 3 7 65 Kd(nM) 10000 850 360 3 0.4 40n7 2'Fluro SELEX SELEX Total Round RNA pm oIe 5 677 659 499 187 179 89 19 17 19 18 99 22 89 92 Total Protein pm ole s 100 100 100 20 20 10 2 2 2 2 12.5 2.5 5 5 Temp, Time, Vol.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

37 0

C.

10111 101±1 10111 I01±1 10111 10111 10111I 10111 10111 10111 5011 50±1 5011 5011 1.8 5.8 9.6 4.3 19.7 17.7 17.3 78.9 36.5 14.1 60.3 90.1 53.2 92.2 Bound LS-Rg Sites 5mM

EDTA

Eluted 0.3 0.9 1.9 2.2 2.0 1.8 10.4 4.1 2.3 7.7 10.4 5.0 Sienal/ Kd(nM) Bkgnd

EDTA

10000 1024 240 0.9 0.3 0.1 WO 96/40703 WO 9640703PCTIUS96/09455 106 Table 15 (Page 2) '~An7 Primer Comnetition Counter-SELEX 30n7 Primer COMDetition Counter-,qELEX SELEX Total Round RNA pm ole s Total Protein pm ole s Temp.

T im e.

VOL

Bound 5mM LS-Rg EDTA Sites Eluted 168 189 185 100 104 37 0 C. 15' 37 0 C. 15' 37 0 C- 15' 37 0 C. 15' 37 0 C. 15' 37 0 C. 15' 1004 10041 10041 loOgi 10041 2.1 15.4 9.2 44.0 29.0 36.0 0.25 1.62 0.99 2.33 1.43 1.70

EDTA

Signall Bkgnd 6 119 66 6 43 24 Kd(nM) 2 0.3 0.4 r, CVIr rv Oynl Primer~ OUiIMLI ill11I ul A SELEX Total Total Round RNA Protein pmoles pmoles Temp, Time, Vol.

Bound LS-Rg Sites 155 184 117 93 93 94 37 0 C. 15' 37 0 C. 15' 37*C. 15' 37 0 C. 15' 37 0 C. 15' 37 0 C. 15' MA~Il 100o±1 100J4d MA0I1 lOOA' 100 p.

1.9 26.8 12.9 46.0 37.0 42.0 5mM

EDTA

Eluted 0.25 2.92 2.21 2.43 2.00 2.25

EDTA

Sign all Bkgnd 172 78 3 52 15 Kd(nM) 2 0.2 0.06 Tabl 16~ L-selectin 21F Ligands Sequences Ligand Sequence SEQ ID No, Family la LF1518 gggaggacgau gcggG LF1817 gggaggacgaugc ggUG LF1813 gggaggacgaugcggUUAAU

UCAGU

LF1822 gggaggacgaugcggCU UAGAG LF1514 gggaggacgaugc ggUAC LF1529 gggaggacgaugcg gACC LF1527(2) gggaggacgaugcg gACC LF1536(2) gggaAgacgaugc ggCG LF1614 gggaggacgaugcggCA AACAAG LF1625 gggaggacgaugcggUA

GUAAGU

LF1728 gggaggacgaugc ggAGA LF1729 gggaggacgaugc ggUG LF1815 gggaggacgaugc ggCCAUCGGU LF1834 gggaggacgaugcggCCAUC

GGUJ

LF15O8 gggaggacgaugcg gGACC LF1828 gggaggacgaugcgg ACGUAGCU LF1807 gggaggacgaugc ggAGU LF1825 gggaAgacgaugcgg AAUGCU LF1855 gggaggacgaugG ggAGU LF1811 gggaggacgaugcgg UAAUCU LF1626 gggaggacgaugcgg CAAUGUCh LF1808(3) gggaggacgaugc ggCAUGGU LF1719(2)* gggaggacgaugcgg GAUG LF1619 gggaggacgaugcggU AAUUGU LF1620 gggaagacgaugcggCUACUA

GUGU

LF1756 gggaggacgaugcggA CUGA LF1629 gggaggacgaugcgg UGGUGUGU LF1821 gggaggacgaugcggUG GUGUGU LF1513 gggaggacgaugcg gGCU LF1615 gggaggacgaugc ggCGUAA

CAAAUUG

CUUAAAC

CUCAAAC

CUCAAAC

CUCAAAU

CUCAAAU

CUCAAAU

CUCAAAU

CUCAAAU

CUCAAAU

CUCAAAU

CUCAAAU

CUUGGGC

CUUGGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUAGGC

CUUUGGC

CUUUGGC

CUUCAGC

CUUCAGC

CAUGCG UU-UU-- AACGCG UGAAU-- GGUGCG UUUAU-- GGUGUG ACUUU-- UGCGUG UU-UU-- AACGUG UCUUU-- AGCGUG CAUUU-- AAUGCG UUAAU-- GACGUG UUUUU-- GUUGCG UUUUU-- GGUGUG UU-UU-- GAUGCG UUUCU-- AACGCG UU-UU-- AACGCG UU-UU-- AACGUG UU-UU-- AAUGCG UAUUU-- AG.CGCG UU-UU-- AGCGCG UUAAU-- AGCGCG UU-UU-- AUCGCG UUAAU-- CACGCG UUAAU-- GACGCG UUUAUAU GCCGUG UU-UU-- GCCGUG UU-AU-- GGAGUG UUUAU-- UGCGCG CACUU-- ACCGCG UAUUUU- ACCGCG UA-UU-- AACGUG UU-AU-- GGUGUG UUAAU--

CGAGUG

CGAGUU

CGAGCC

CAAGCC

CAAGCA

CAAGUU

CAAGCU

CGAAUU

CAAGUC

CGAAAC

CAAGCC

CGAAUC

CGAGUU

CGAGUU

CAAGUU

CGAAUU

CGAGCU

CGAGCU

CGAGCU

CGAGAU

CGAGCG

CGAGUC

CAAGGC

CAAGGC

CAAUCC

CUUGC UcagacGacucgcccga CAUC CACUCCUCCU cagacgacucgcccga ACUGA UcwgacgacucgcccgaA CUCUA UGCCcagacgacucgcccga GUAUc agacgacucgcccga GGUc agacgacucgcccga GGUc agacgacucgcccga CGCC cagacgacucgcccga CUUGUU GUcagacgacucgcccga ACUUAC AUcaGacgacucgcccga UCUCC cagUcgacucgcccga CACC cAgacgacucgcccga GG ACCUAUGGUc agacgacucgcccga aCC UACAUcagacgacucgcccga GGUc agacgacucgcccga AGCUGUGU cagacgacucgcccga ACUCC AUCGCCAGUcagacgacucgcccga AGCACAUCCUcagacgacucgcccga ACUCC AUCGCCAGUcagacgacucgcccga AGAUCACCGU cagacgacucgcccga UGACUGU cagacgacucgcccgag ACCAUGCU cagacgacucgcccga CAUc agacgacucgcccga ACAAUU UCCCUcagacgacucgcccga ACAC allcagacgacucgcccga 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 CGAGCA UcaG acgacucgcccga CGAGGU ACACAUca gacgacucgccga CUCGAG GUACAC AUcagacgacucgcccga CAAGUU AGCCc agacgacucgcccga CAAGCC UUACGCC AUCUcagacgacucgcccga Table 16 (Page 2) Ligand Sequence SEQ ID No, Family la (continued) LF1521(2) gaggacgaugc ggGCU CUUAAGC LF1651 gggaggacga ugcggU CUCAAGC LF1830 gggaggacgaugcggAA AUCU CUUAAGC LF1523 gggaggacgaugc ggUU CUtJAAGC LF1708** gggaggacgaugc ggAU CUUAAGC LFl1851 ACAGCUGAUGACCAUGAUUACGCCAAG

CUUAAGC

LF1610(3)** gggaggac gaugcggAGGGU CUUAAGC LF1712 gggaggacgaugc ggGAU CUUAAGC LF1613(3) gggaggacgaugcggUGC UAUU CUUAAGC AACGUG UU-AUaAUGCG UUUAU-- AGCGUG UAAAU- AGCGCG UCAAU-- AGCGCG UCAAU-- AGCGCG UU-UU-- AGUGUG AUAAU- AGUGCG UUAUU-- GGCGUG UUUUU-- GGCGCG AUUUU-- GUCGUG UUUUU-- GACGUG CAUUU-- GGCGUG UAAAU-- UGUGCG UU-UU-- LF1735 LF1731 LF1853 LF1816 LF1622 (3 LF1725 LF1 632 LF1 856 LF1631 LF1730 LF1 852 LF 1653 LF1554 LF 1722 gggaggac gaugcggU gggaggacgaugcg gCCU ggga ggacgaugcggAUACCACCU gggaggacgAugcggUGCUA

UU

*gggaggacgaugcggA ACGACJ gggaggacgaugc ggCU gggaggacgaugcggAG UCWCU gggaggacGaugcggUCUAC GGUCU gggaggacgaugc ggUdAUUU

CUUAAGC

CUUAAGC

CUUAAGC

CUUAAGC

CUUAAGC

CUCAUUU

CUCcacC

CUCUGGC

CyUAAUC

CAAGUJ

CGAAUU

CAAGCU

CGAGCU

CGAGCU

CGAGCU

CAAACU

CGAACU

CAAGCC

CGAGCC

CAAGCU

CAAGUC

CAAGCU

CGAACA

CGAGCU

CAAGCU

CkAACC

CUAUCU

CkAAAC

CAAGCU

CAAGCU

CAAACU

CGAGCU

AGCCc agacgacucgcccga ACCGUA CGCCUCCGUcagacgacucgcccga AGAU CUUCGUcAgacgacucgcccga AACC cagacgacucgcccga AACC cagacgacucgcccgag CAUGUUGGUcagacgacucgcccga ACUCUCCGUGUc agacgacucgcccga AUCCc agacgacucgcccga AAUA UCAUcagacgacucgcccga ACCGCAUCCUC CGUGcaGacgacucgcccga GGUc agacgacucgcccga AGAUGGucagacgacUcgcccga AG AUCAUCGUcagacgacucgcccga AGUCGU AACUcagacgacucgcccga AGCC cagacgacucgcccga MnUG CCUcagacGacucgcccga AGAUCG cagacgacucgcccga mAAUkAUC CUcagacgacucgcccga AGCC Ucagacgacucgcccga AAGUUCC ACCUcagacgacucgcccga AUUGU UGUcagacgacucgcccga AUCGUc agacgacucgcccga AACC cagacgacucgcccga 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 371 372 366 367 gggaggacgaugc ggaU CgCAAUmU gggaggacgaugc ggAACUU CUUAGGC gggaggacgaugcggC ACAAU CUUCGGC gggaggacgaugc ggCGGU CUUAAGC gggaggacgaugc ggUU CUUAAGC wGCGCG AkCGUG

GGUGCG

hGAGCG GUwGCG

AGCGUG

AGCGUG

AGUGUG

AGCGCG

UAAAU- UkUUAAU UAAAU- UUUAU- UU-cU-- CUAGU-

CAAGAU-

UCAAU- UCAAU- Truncates LF1 51 4T1 LF1514T2 LF1514T4 LF1807T5

CUCAAAU

ggUAC CUCAAAU ggAGU CUUAGGC

UGCGUG

UGCGUG

UGCGUG

AGCGCG

UU-UU--

UU-UU- UU-UU- UU-UU-

CAAGCA

CAAGCA

CAAGCA GUAUc CGAGCU ACUCC Table 16 (ae3 (Page 3) Ligand Sequence SEQ ID Family lb LF15l1(4 gggaggacgaugcgg UGGUU LF1753 gggaggac gaugc LF1524 gggaggacg augc LF1810 g ggaggacgaugc LF1621 *gggaggacgaugcggUCAA AGUGAAG LF1826(2)* gggaggacgaugcgg GGAG LF1713 gggaggacgaugcgg GAGG LF1520 gggaggacgaugcg gAUCG LF1552 gggaggacgaugcggA GACc

CUAG

ggAA ggAA ggAG

AAUG

AAUG

AAUG

AUU

CUA

GCACGUG

ACAUGUG

GGCCGUG

GAUGGUG

GACaGCG

GCCAGCG

GACwGCG

UCAUGCG

AGmGsG UU-UU- UU-UU- UUAAU UUCAU- UU-UU- UUUAU- UAUAU- UUUUU- UksUUUU

CAAGUGU

CGAAUGU

CAAGGCU

CAAGAUU

CGAGUU

CGAGGU

CGAGUUG

CGAGUGA

CAAsCU AAUca gacgacucgcccga gCUC UCCUCCCCAAACAACyCCCCCAA GCAAU AAAUCAUCCUCCC cagacgacucgcccga GCUCGUUCUUU ACUGCGUUcagacgacucgcccga GCUUCACU cagacGacucgcccga GCUCCGUUAACCGG cAgacgacucgcccga CCUc agacgacucgcccga CGAUc agacgacucgcccga GGUc wgacgacucgcccga Family lc LF1618 (2) LF1528 (3) LF 1718 LF1623 LF155S7 gggaggacgaugcgg UUAGCCUACACUCUAGGUUCAG gggaggacgaugcgg UUAGGUCAAUGAUCUUAG gggaggacga. ugcggA CGUGUG UAUCrAr gggaggacGaugcgg ACAGGGUUCUUAG GCGGAG gggaggacgaugcgg CGAUUUCCAC AGUUUG

UU-UU--

UU-UU- UU-UU- UG-UU-

UCUUAUU

CGAAUCUUCCACCG cWgacgacucgcccga CGAUUCGU cagacgacucgcccga CCGCUG UUUGUG cagacgacucgcccga CAUCAA UCCAACCAUGU cagacgacucgcccga CCGCAU AU cagacgacucgcccga Family 1 (Unclassified) LF1707 LF 1512 LF1535 (2) LF1711 LF 1517 gggaggacgaugcgg AUAyUCAgCUyGUGUk gggaggacgaugc ggCACACGUG gggaggacgaugc ggCAAUGUG gggaAgacg augcggUG gggaggacgau gcggUG

UU-UU--

UU-UU--

UUUCU- UUGAU- UUUGU- CdAUCUUCCC cagacgacucgcccga CAAGUGUGCU CCUGGGAU cagacgacucgcccga CAAAUUGCU UUCUCCCUU cagacgacucgcccga CAAUG AAUGUCCUCCUCCUACCC cagacgacucgcccga CAAUGU CAUGAUUAGUUUUCCCA cagacgacucgcccga 356 357 358 364 365 Table 16 (Page 4) Ligand Family 2 LF1627 (2) LF1724 (2) LF1652 (2) LF1519 LF1608 Family 3 Sequence SEQ ID gggaggacgaugc gggaggacgaugc gggaggacgaugc gggaggacgaugc gggaggacgaugc ggAUACUACCGUGCG AACaCUAAG UCCCGUCUGUCCACUCCU cagacgacucgcccga ggAUaCUA-UGUGCG UUCACUAAG UCCCGUC-GUCCCCU cagacgacucgcccga ggGUACUA UGUACG AUCaCUAAG CCCCAUCACCCUUCUCACU cagacnacucgcccga ggUUACUA UGUACA UUUACUAAG ACCCAACGU cagacgacucgcccga ggUUwCUA UGUWCGCCUUACUAAGUACCCGUCGACUGUCCCAU cagacgacucgcccga 359 360 361 362 363 LF1710 LF1829 LF1 509 gggaggacgaugcgg AAUGrCCCGUUACCAwCAAUGCGCCUCdUUGMCCCCAAACAACyCCCCCAA gacgaugcgg AAUyUCGUGyUAcGCGUyyyCUAUCCAAUCUACCCCMUCUCCAAU cagacgacyc gggaggacgaugcgg CGCUUACAAUAAUUCUCCCUGAGUACAGCucag acgacucgcccga 368 369 370 orphans LF 150'7 LF1516 (2) LF1530(2) LF1835(4*) monster LF1522 LF 1727 LF 1510 LF1 715 LF1809 LF1533 LF1720 gggaggacgaugcgg gggaggacgaugcgg gggaggacgaugcgg gggaggacnaugcgg gggaggacgaugcgg gggaggacgAugcgg gggaggacgaugcgg gggaggacgaugcgg gggaggacgaugcgg AggaggacGaugcgg Aggaagacgaugcgg UCAUUAACCAAGAUAUGCGAAUCACCUCCU cagacgacucgcccga UCAUUCUCUAAAAAAGUAUUCCGUACCUCCa cagacgacucgcccga GUGAUCUUUUAUGCUCCUCUUGUUUCCUGU cagacgacucgcccga UCUAGGCaUCGCUAUUCUUUACUGAUAUAAUUACUCCCCU cagacgacucgcccga AGUwwGCNCGGUCCAGUCACAUCCwAUCCC cagacGacucgcccga CUCUCAUAUkGwGUrUUyUUCmfUUCs rGGCUCAAACAAyyCCCCCAA CUUGUUAGUUAAACUCGAGUCUCCACCCCU cagacgacucgcccga UCUCUwCUvACvUGUrUUCACAUUUUCGCyUCAAACAACyCCCCCAA UUrACAAUGrssCUCrCCUUCCCwGGUCCU cagacgacucgcccga UUAUCUGAArCwUGCGUAAmCUArUGUsAAAsUGCAACrA cRaacaacYcScccaa UUCGAUUUAUUUGUGUCAUUGUUCUUCCAU cagacgacucgcccga GUGAUGACAUGGAUUACGC cagacgacucgcccga WO 96/40703 PCT1US96/09455 M1 Table 17 2' Fluoro L-seilectin SELEXes: Full Length Transcribed Ligands: Protein and Lymphocyte Binding Affinity L-selectin# Lymnphocytes# LIGAND SEO ID NO Kd (nM) Kd (nM) LF1508 307 LF1511 342 0.48 LF1512 357 315 LF1513 321 0.16 4 LF1514 297 0.13 0.8 LF1516 374 1.3* LF1518 293 0.42 LF1520 339 LF1521 323 0.25* LF1523 326 0.25 LF1524 344 2.1* LF1527 299 0.32 LF1528 352 LF1529 298 0.6 LF1535 358 LF1536 300 0.22* LF1610 329 0.53 LF1613 331 0.034 0.2 LF1614 301 0.17 LF1615 322 0.32 LF1618 351 9.6 LF1707 356 0. 16* WO 96/40703 PCTIUS96/09455 112 Table 17 (Page 2) L-selectin# Lymphocytes# LIGAND SEO ID NO Kd (nM) Kd (nM) LF1708 327 LF1712. 330 0.065* LF1713 338 0.22* LF1718 353 6.4* LF1807 309 0.034 LF1808 314 0.6 LF1810 345 8.1* LF1811 312 0.19 LF1 815 305 0. 18* LF1816 335 LF1817 294 2.3* 40N7 NX280 1.6 3 Nitrocellulose flter partitioning 37 0

C;

*designate soluble L-selectin, others LS-Rg; indicates binding was undetectable Flow cytomnetry competition room temperature; Table 18 P-SELECTIN 2'F RNA SELEX SELEX RNA Load Ro~und (PMol) PS-Rg Bead (pmoll Volume Rnd 1 Rnd 2 Rnd 3 Rnd 4 Rnd 5 Rnd 6 Rnd 7 Rnd 8 Rnd 9 Rnd 10 Rnd 11 Rnd 12 Rnd 13 320 510 200 200 25 25 25 50 50 10 5 5 0.1 200 100 40 40 8 5 5 5 5 5 1 1 1 0.5 1 0.2 0.1 0.01 10 10 10 10 10 10 10 10 10 10 10 10 100 10 250 10 250 250 Totl %RN Volume eluted mM

EDTA

125 III 1.4 125 ttl 1.8 125 ttd 1.7 125 ,iI 2.3 125 pi 1.3 125 VI 1.2 125 pI 0.9 125 pi 0.8 125 pI 4.0 125 [d 4.6 125 [d 4.5 125 p1 5.3 250 tt1 2.8 500 pi 5.6 500 0II 10 500 p 1 l 14.2 500 p1l 4.5 500 tti 2.6 Signal to

EDTA

8 9 5 15 4 3 3 2 8 16 6 28 6 20 11 15 4 2 eluted 8.3 3.5 2.6 3.0 0.8 0.6 0.15 0.0 1.0 0.4 0.2 0.05 0.3 0.2 0.4 0.6 0.8 Signal to

EDTA

40 30 12 13 8 3 1.5 1 4.3 6.7 2.3 1.5 2 5 2 3 2

N~D

on column 0.7 0.6 0.3 0.1 0.3 0.7 0.3 0.4 0.5 0.3 1.4 1 .2 0.8 1.2 2.5 13 4.7 3.6 Kd (nM) 2500 1200 280-900 13 0.1-2 0. 02-20 Table 19o P-Selectin 2'-F RNA Ligazids SEQ ID Ligand SequenceNO Family 1 PF37 3(6) gggagacaagaauaaacgcucaaCGAAUCAGUAAACAUAACACCAUGAAACAUAAAUAGCACGCGAGACGUCuucgacaggaggcucacaacaggc 199 PF424 gggagacaagaauaaacgcucaaCGAGUUCACAUGGGAGCAAUCUCCGAAUAAACAACACGCKAKCGCAAAuucgacaggaggcucacaacaggc 200 PF4 12 gggagacaagaauaaacgcucaaCGACCACAAUACAAACUCGUAUGGAACACGCGAGCGACAGUGACGCAUUuucgacaggaggcucacaacaggc 201 PF4 22 gggagacaagaauaaacgcucaaCGUCAAGCCAGAAUCCGGAACACGCGAGAAAACAAAUCAACGACCAAUCGAuucgacaggaggcucacaaaggc 202 PF4 26 gggagacaagaauaaacNcucaaCGACCACAAUAACCGGAAAUCCCCGCGGUUACGGAACACGCGAACAUGAAuucgacaggaggcucacaacaggc 203 PF3 98 gggagacaagaauaaacgcucaaCGAACCACGGGGAAAUCCACCAGUAACACGCGAGGCAAACAGACCCUCuucgacaggaggcucacaacaggc 204 PF380 gggagacaagaauaaacgcucaaCGAGCAAAAGUACUCA CGGGACCAGGAGAUCAGCAACACGCGAGACGAAAuucgacaggaggcucacaacaggc 205 PF377 gggagacaagaauaaacgcucaaCGAGCCAGGAACAUCGACGUCAGCAAACGCGAGCGCAACCAGUAACACCuucgacaggaggcucacaacaggc 206 PF387 gggagacaagaauaaacgcucaaCGCACCAGGAACAACGAGAACCAUCAGUAAACGCGAGCGAUJGCAUGuucgacaggaggcucacaacaggc 207 PF3 83 gggagacaagaauaaacgcucaaCGCACCAGGAACAACAAGAACCAUCAGUAAGCGCGAGCGAUUGCAUAuucgacaggaggcucacaacaggc 208 PF3 95 gggcaauacccaGGAGACAUCACAGACCGACCCACGAGuugcgaguaacg 209 PF416 gggagacaagaauaaacgcucaaCAGUUCACUCAACCGGCACCAGACUACGAUCAGCAUUGGCGAGUGAACACuucgacaggaggcucacaacaggc 210 PF388 gggagacaagaauaaacgcucaaCUGGCAACGGGAUAACAACAAAUGU CACCAGCACUAGCGAGACGGAAGGuucgacaggaggcucacaacaggc 211- Family 1 Truncates PF3 73 si CUCAACGAAUCAGUAAACAUAACACCAUGAAACAUAAAUAGCACGCGAG 220 PF 424 si CUCAACGAGUUCACAUGGGAGCAAUCUCCGAAUAAACAACACGCGAG 221 PF3981 CUCAACGAACCACGGGGAAAUCCACCAGUAACACGCGAG 222 PF3 77 si CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 223 PF3 77 s2 CGCUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAGCG 224 PF377 LI CUCAACGAGCCAGGACUACGAUCAGCAAACGCGAG 225 PF3 87s 1 CUCAACGCACCAGGAACAACGAGAACCAUCAGUAAACGCGAG 226 PF3 83 si CUCACGCACCAGGAACAACAAGAACCAUCAGUAAGCGCGAG 227 PF4 16s2 CACUCAACCGGCACCAGACUACGAUCAGCAUUGGCGAGUG 228 PF4 22 si GAAUCCGGAACACGCGAGAAAACAAAUCAACGACCAAUCGAUUCG 229 Table 19 (Page SEQ ID Ligand SequenceNO 2'OMty Substituted Truncates PF3 77M1 CUCAACGAGCCAGQAACA&UCQACGUCAGCAAACGCGAG 230 PF3 772 CUCAACGAGCCAQ2aAACAUCGACQUCAgCA&ACGCGAG 231 PF3 77M3 CUCAACGAGCCAGQAACAUCGACGUCA&GCACGCQG 232 PF3 77M4 CUCAACGAGCCA~AACAUCQAUUCACAACGCQMG 233 PF3 77M5 CUCAACgAGCCAGQAACAUCGACGUCAGCAAACGCGAG 234 PF 377 M6 CUCAACGAGCCAGGAACAUCGACQOUCAGCAAACGCGAG 235 Family 2 PF37 8(8) gggagacaagaauaaacgcucaaCGAUGAGCGUGACCGAAGCUAUAAUCAGGUCGAUUCACCAAGCAAUCUUAuucgacaggaggcucacaacaggc 212 Family 3 PF381 gggagacaagaauaaacgcucaaAGGAUCACACAAACAUCGGUCAAUAAAUAAGUAUUGAUAGCGGGGAUAuucgacaggaggcucacaacaggc 213 Family 4 PF411 gggcaauacccaACCAAUUGGUCACAGUUCAGACAAuuggagucagcg 214 k-n Family PF396 gggagacaagaauaaacgcucaaGCGGUCAGAAACAAUAGCUGGAUACAUACCGCGCAUCCGCUGGCGAUAuucgacaggaggcucacaacaggc 215 orphans PF3 86 gggagacaagaauaaacgcucaaACAAGAGAGUCAAACCAAGUGAGAUCAGAGCGUUUAGCGCGGAAAGCACAuucgacaggaggcucacaacaggc 216 PF3 82 gggagacaagaauaaacgcucaaACUCGACUAGUAAUCACCCUAGCAJAAAUCUCCUCGAGCACAGACGAUAuucgacaggaggcucacaacaggc 217 PF404 gggagacaagaauaaacgcucaaUCAGCAGUAAGCGAUCCUAUAAAGAUCAACUAGCCAAA3AUGACUUAuucgacaggaggcucacaacaggc 218 PF417 gggagacaagaauaaacgcucaaAAAGACGUAUUCGAUtJCGAAACGAGAAAGACUUCAAGUGAGCCCGCAouucgacaggaggcucacaacaggc 219 WO 96/40703 PTU9/95 PCTIUS96/09455 Dissociation Table Constants and Specificity of 2'F RNA Ligands to P-Selectin Ligand PF373 PF377 PF378 PF380 PF381 PF386 PF387 PF388 PF395 PF396 PF398 PF404 PF411 P F41 2 P F41 6 PF41 7 PF422 PF424 Kd (PS-Rg) 49.5 pM 18.5 pM 51.5 pM 74.5 pM 16.5 pM 45.5 pM 16 pM 90 pM 26 pM 24 pM 46 pM 47.5 pM 13 pM 450 pM 63 pM 69 pM 172 pM 36.5 pM S LeX (I C50) Kd (ES-Rg) 3 g±M 2.3 gM Kd (LS-Rg) 3 g~M 3 g.M Tm(oC) 3 nM 4 nM 1 M 2 nM 3 nM 53oC SEQ ID

NO.

1 99 206 212 205 213 216 207 211 209 215 204 218 214 201 210 219 202 200 Table 21 Boundary Results for 21F RNA Ligands to P-Selectin FAMILY 1 SEQ ID Kd(pM) Clone# 56 178 63

ND

PF373s1 PF424s1 PF398S1 PF38Osl cucaaCGAAUCAG UA AACAUAACACCAUGAAACA cucaaCGAGUUCACAUG GGAGCAAUCUCCGAA -cucaaCGAACCAC GG GGAAAUCCA cucaaCGAGCAAAAGUACUCACGGGACCAGGAGA PF377s1 PF377s2 PF412 63 PF387s1 10000 PF383s1 PF388 150 PF416s1 PF3 95 PF426 1000 PF422sl cg cg cg acg cg

UCA

gggagacaagaauaaacg -cucaaCGAGCCAG cucaaCGAGCCAG cucaaCGACCACAA cucaaCGCACCAG cucaaCGCACCAG cucaaCUGGCAAC CjUQAACQGGQACCA cucaaCGAGCAAG cucaaACACAA

AUCAACGACCAAUC

ACAUCGACG

ACAUCGACG

CAAACUCG

ACAACGAGAACCA

ACAACAAGAACCA

GAUAACAACAAAUGUCA

CUACGA

UAAAUAGCAC-CGAG

UAAACAACA-CCGAG

9CAGtJAACACGCGAG

UCAGCAACACGCGAG

UCACAAA CGGA UCAGCAAA CGCGAG

UAUGGAACACGCGAG

UCAGUAAA CGCGAG UCAGUAAG CQGCGAG CCAGCACU AGCGAG UCAGCAUU GGCGAG ACGAAAuucg

CG

CG

CG

CG

CG

ACG

UG

CA

CA

AAAACAA

ACGAAUACAAACCAGGAAACEPiGCAACACGCGAG ACCGGAAAUCCCCGCGGU IIACMAACAMC-GA uucg3l 5 GAAUCCGGAACACGCGAG PF378 PF381 PF396 PF411 PF386 PF417 FAMILY 2 agaatiaaacgcucaaCGAUGAGCGUGACCGAAGCUAUAAUCAGGUCGAUUCACCAAGCAAUCUUAuucg FAMILY 3 acacucaaAGGAUCACACAAACAUCG-IAAVAAUAAGUAUUGAUAGCG FAMILY 4 cucaaGCGGUCAGAAACAAUAGCUGGAUACAUA CCGCgCAUCCGCUGGGCG FAMILY ACCAUCUAGAGCUUCGAACCAUGGUAUACAAGGGAACACAAAAuuc gcggaggcucca 242 243 244 245

ORPHANS

gggagacaagauaaacgcucaaACAAGAGAGUCAAACCAAGUGAGA1JCAGAGCGUUUAGCGCGGjAAGCACAuucgacaggaggcucacaacaggc 246 gggagacaagaauaaacgcucaaAAGACGUAUUCGAUUCGAcQGAAAGAC UUCAAGUGAGCCCGCAGuucgacaggaggcuca 247 WO 96/40703 PTU9195 PCT[US96/09455 118 Table 22 Dissociation Constants and Specificity of Truncated 2'F RNA Ligands to P-Selectin Kd S LeX Kd (PS-Rg) (IC50) (ES-Rg) Kd (LS-Ra) Tm

(OCI

B~se~ SEQ ID Ligand Bases

NO

PF373s1 PF377s1 P F377s2 PF383s1 PF387s1 PF398s1 PF41 6s2 PF422sl PF377s1 B PF377s1 B:SA PF377s1 F PF377s1 5'NH2 PF3771 PF377t3' 56 pM 60 pM 45 pM 10000 pM 63 pM 178 pM 150 pM 1000 pM 65 pM 30 pM 60 pM 125 pM 220 pM 30 pM 120 pM 1700 pM 900 pM 1700 pM 60 pM 250 pM 3 nM 2 nM 4 nM 25 nM 2 nM 2 nM 3 nM 8 nM 3 p.M 3 p.M 3 p.M 3 p.M 3 jiM p.M 3 jiM 3 gM >3p.M >3p.M 590C 3nM >3p.M >3j±M 3 nM 220 223 224 227 226 222 228 229 223 223 223 223 225 223 230 231 232 233 234 235 2 nM 4 nM 2 nM >3p.M >3p.M PF377M 1 PF377M2 PF377M3 PF377M4 P F377M5 PF377M6 3gpM 10 nM >3 gM 2 nM 3 IM WO 96/40703 PCTIUS96/09455 119 Table 23 2'OMe Substitution of 27F RNA Ligands to P-Selectin Punine Unmixed Std. Dev. Mixed Mixed Predicted Actual Position Ratio 40 pM 200 pM Pref. Pref.

4 7 8 12 13 14 16 18 21 22 24 27 28 31 32 34 1.07 1.00 1.00 1.00 0.83 0.90 0.73 0.63 0.67 0.60 0.87 0.72 0.70 0.83 0.69 0.90 0.92 1.10 0.93 0.12 1.00 0.13 0.20 0.12 0.17 0.15 0.15 0.1'0 0.10 0.30 0.16 0.16 0.12 0.09 0.00 0.16 0.06 0.06 0.3 0.4 1.2 2.3 0.4 0.8 0.8 0.8 0.5 0.7 0.5 0.7 0.6 1.3 0.6 0.8 1.2 0.5 0.7 0.4 0.7 1 .5 1 .3 0.5 0.8 0.9 1 .3 0.7 0.7 0.7 0.8 0.8 1.5 1.0 1.0 1.5 2'-OH 2'-OH 2'-0-Me 2'-0-Me 2'-OH neutral neutral 2s-0-Me neutral neutral neutral neutral neutral 2'-0-Me 2'-O-Me neutral 2'-0-Me untested untested 2'-O-Me untested 2'-O-Me untested 2'-O-Me untested untested 0.9 2'-OH Table 24 P-Selectin 2'NH 2 RNA SELEX SELEX EINALoad Blun4# Lpmol Rnd 1 Rnd 2 Rnd 3 Rnd 4 Rnd 5 Rnd 6 Rnd 7 Rnd 8 Rnd 9 Rnd 10 Rnd 11 Rnd 12 330 30 0 550 500 365 500 50 50 10 200 100 100 100 100 50 5 5 5 1 0.2 0.1 0.1 0.1 0.1 Bead Total

RNA

Volume Yrnnm 2W~uig mm

EDTA

10 111 125 ji1 0.0 10 PI 100 PI 0.-8 10 P1 125 gI 0.6 10 111 125 pI 1.0 10 A1 125 pI 1.5 10 PI 125 pf 1.9 10 A1 125 p1 1.1 10 PI 125 pi 1.8 10 P1 125 p1 3.6 10 A1 125 p1 3.3 10 111 500 pi 2.5 10 A1 500 pl 2.0 250 pi 500 p1 1.5 10 P1 500 Al 4.1 250 g1 500 pti 3.1 Snal to 5 mm

~I

8 21 33 30 22 5 7 7 5 3 2 2 5 2

EDTA

1.3 0.3 0.2 0.8 1.6 -0.9 0.4 0.05 0.0 0.1 0.0 0.0 0.0 0.2 0.2 Sigqnal to 6.5 2.7 8 10 32 17 2.3 1.8 <1 2 <1 <1 2 1 onYoum 0.2 0.6 0.1 0.4 0.4 0.3 1.2 0.6 0.6 1.2 0.3 12.0 3.2 14.0 6350 1900 470 103 31 0.2-6 .Table P-Selectin 21NH 2 RNA Ligands Ligand PA341 (7) PA3 50 PA4 66 PA4 73 PA477 (3) PA328 (3) SEQ ID

NO.

Sequence f amily I gggagacaagaauaaacgcucaaGCCCCAAACGCAAGCGAGCAUCCGCAACAGGGAAGAAGACAGACGAAUGAuucgacaggaggcucacaacaggc gggagac aagaauaaacgcucaaGCCCCAAACGCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGACGAUUGAuucgac aggaggcucacaacaggc gggagacaagaaauaaacncucaaGCCCCAAACGCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGAUGAAUGAuucgacaggaggcucacaacaggc gggagacaagaauaaacncucaaGCCCCAA GCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGACGAGUGAuucgacaggaggcucacaacaggc gggagacaagaauaaacncucaaGCCCCAAaCGCAAGUG AGCAUCCGCAACAGGGAAGAAGACAGACGAAUGAuucgacaggaggcucacaacaggc gggagacaagaauaaacgcucaaGCAAAAGGCGUAAAUACACC UCCGCAACUGGGAAGAAGACGCAGGGACGGuucgacaggNggcucacaacaggc f amily 2 PA337 gggagacaagaauaaacgcucaaACAGCUACAAGUGGGACAACAGGGUACAGCGGAGAGAAACAUCCAAACAAGuucgacaggaggcucacaacaggc family 3 PA4 48 gggagacaagaauaaacgcucaaAUCAACUAAACAACGCAGUCACGAGAACGACCGGKCUGACUCCGAAAG uucgacaggaggcucacaacaggc others PA3 25 gggagacaagaauaaacgcucaaACGAGAGCACCAAGGCAACAGAUGCAGAAGAAGUGUGCGCGCGCGAAA uucgacaggaggcucacaacaggc PA3 27 gggagacaagaauaaacgcucaaUAAGACAACGAACAGACAGAAGCGAAAAAGcGGGCGCCGAGCAACAACAAAuucgacaggaggcucacaacaggc PA4 46 PA3 13 PA3 36 PA301 PA 305 PA 309 PA3 15 PA3 18 PA319 PA320 PA3 21 PA3 24 PA3 29 PA3 30 PA332 PA335 PA336 gggagacaagaauaaacgcucaaCGUGUACCACAACAGUUCCACG GAAGCUGGAAUAGGACGCAGAGGAA gggagac aaga auaaacgcucaaACAAAAUUWUGGUGGGCCCCGcAACMGGGRGGRAGRCCGUUGAAGGC gggagac aa gaauaaacgcucaaGAUCAUAACGAGAGGAGAGGGAGAACUACACGCGCGCGAGGAAAGAG gggagacaagaauaaacgcucaaACACAAAUCGGGCAGGGACUGGGUUGGGCACGGCAGGGCGCC gggagacaagaauaaacgcucaaGUGGGCUCGGGCCGGAUGUCUACGGGUGUGAAGAAACCCCJAGGGCAGGG gggagacaagaauaaacgcucaaGAUCAGCGGAACUAAGAAAUGGAAGGCUAAGCACCGGGAUCGGGAGAA gggagacaagaauaaacgcucaaUAACAAAGCAGCAAAGUACCAGAGGAGAGUUGGCAGGGUUUAGGCAGC gggagaca -gaauaaacgcucaaAGACCAAGGGACAGCAGCGGGGAAAAACAGAUCACAGCUGUAAGAGGGC gggagacaagaauaaacgcucaaAGUCGGGGAUAGAAACACACUAAGAAGUGCAUCAGGUAGGAGAUAA gggagacaagaauaaacgcucaaGAGUAUCACACAAACCGGCACGGACUAAGCAGAAGGAGGUACGGAAGA gggagacaagaauaaacNcucaaCGAAAUAGAAGGAACAGAAGAAUGGBGAWGNGGGAAAUgGCAACGAA gggagacaaga auaaacgcucaaACGAGACCCUGGAUACGAGGCUGAGGGAAAGGGAGMMM~RRAMCUARRCKC gggagac aagaauaaacgcucaaGAAGGAUACUUAGGACUACGUGGGAUGGGAUGAAAUGGGAGAACGGGAG gggagacaagaauaaac gcucaaAACGCACAAAGUAAGGGACGGGAUGGAUCGCCCUAGGCUGGAAGGGAAC gggagacaagaauaaac gcucaaGGUGAACGGCAGCAAGGCCCAAAACGUAAGGCCGGAAACNGGAGAGGGA gggagac aagaaua aacgcucaaUGAUAUACACGUAAGCACUGAACCAGGCUGAGAUCCAUCAGUGCCCAG3 gggagacaagaauaaacgcuc aaGAUCAUAACGAGAGGAGAGGGAGAACUACACGCGCGCGAGGAAApGAG uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggnggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggnggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggnggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 Table 25 (Page 2) SEQ ID

NO.

Ligand Sequence PA3 38 PA3 39 PA3 42 PA3 49 PA3 51 PA3 52 PA3 53 PA3 54 PA447 PA4 63 PA4 65 PA4 67 PA4 79 gggagacaagaauaaacgcucaaUCAAGUAAGGAGGAAGGGUCGUGACAGAAAAACGAGCAAAAAACGCGAG gggagacaagaauaaacgcucaaAAGGUGCCGGGUUGGAGGGGUAGCAAGAAAUGGCUAGGGCGCASGA gggagacaagaaua aacgcucaaCCAACGCGCACCCCGCAGCAAACGAAAUUGGGGAGACAGGUGCAAGACAG gggagacaagaauaaackcucaaCAAACAAUAUCGGCGCAGGAAAACGUAGAAACGAAAMGGAGCUGCGYGGA gggagacaagaauaaacgcucaaUGAUAGCACAGUGUAUAAGAA-AACGCAACACCGCGCGCGGAAAGAG gggagacaagaauaaacgcucaaGAUCAUCGCAGUAUCGGAAUCGACCCtJCAGUGGGUGACAUGCGGACAAG gggagacaagaauaaacgcucaaGUACCGGGAAGGGAUGAACUGGGAUAUGGGAACGGAGGUCAGAGGCACGA gggagacaagaauaaacgcuc aaGCAAUGGAACGCUAGGAGGGAACAUAAGCAGGGCGAGCGGAGUCGAUAGC gggagac aagaauaaacgcucaaAACAGAACUGAUCGGCGCAGGUUGAUAAAGGGGCAGCGCGAAGAUCACAA gggagac aagaauaaacgcucaaGGGAAACGGAAAGGGACAAGGCGAACAGACGAGAAGUAGACGGAGUAGGA gggagaca agaauaaacgcucaaNNiNGAGGAAGGGCACGCAAGGAAACAAAACACAAAGCAGAAGUAGUAAGA gggagacaagaauaaacgcucaaGUACRCAGUGAGCAGAAGCAGAGAGACUUGGGAUGGGAUGAAAUGGKC gggagacaagaauaaacNcucaaCCGACGUGGACDCGCAUCGGCAUCCAGACCAGGCUGNBCNGCACCASACG uucgacaggaggcucacaacaggc uucgacaggnggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggaggcucacaacaggc uucgacaggagg cucac aa caggc uucgacaggaggcucacaacaggc 278 279 280 281 282 283 284 285 286 287 288 289 290 PCTILJS96/09455 WO 96/40703 123 Table 26 Dissociation Constants and Specificity of 2'NH2 RNA Ligands to P-Selectin Ligand PA301 PA305 PA309 PA3 15 PA318 PA319 PA320 PA321 PA325 PA327 PA328 PA329 PA330 PA335 PA336 PA337 PA338 PA339 PA341 PA342 PA350 PA351 PA352 PA353 PA354 PA447 PA448 PA463 PA465 PA466 PA467 PA473 PA477 Kd 2.5 nM 0.21 pM 0.656 pM 5 nM 2 nM 11 nM 4.5 nM 8 nM 10 nM 13.5 nM 3 nM 4 nM 0.237 nM 10.5 nM 15 nM 4.5 nM 57 nM 13.5 nM 0.44 nM 4 nM 0.06!nM 2 nM 6 nM 9 nM 5 nM 50 nM 5 nM 8 nM 50 nM 0.43 nM 24 nM 0.36 nM 0.57 nM Kd SLeX Kd (4oC) (1C50) (ES-Rq) Kd (LS-Ra) SEQ ID

NO.

264 265 266 267 268 269 270 271 259 260 256 273 274 276 277 257 278 279 251 280 252 282 283 284 285 286 258 287 288 253 289 254 255 3 nM 0.01 nM 2 nM 375 nM 3 jiM WO 96/40703 PCTfUS96/09455 124 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: PARMA, et al.

(ii) TITLE OF INVENTION: HIGH AFFINITY NUCLEIC ACID LIGANDS TO LECTINS (iii) NUMBER OF SEQUENCES: 390 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Swanson Bratschun, L.L.C.

STREET: 8400 E. Prentice Avenue, Suite 200 CITY: Englewood STATE: Colorado COUNTRY: USA ZIP: 80111 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3 1/2 diskette, 1.44 MB COMPUTER: IBM pc compatible OPERATING SYSTEM: MS-DOS SOFTWARE: WordPerfect (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US96/09455

(B)

(C)

(vii)PRIOR

(A)

(B)

(vii)PRIOR

(A)

(B)

FILING DATE: 05 JUNE 1996

CLASSIFICATION:

APPLICATION DATA: APPLICATION NUMBER: 08/479,724 FILING DATE: 07-JUNE-1995 APPLICATION DATA: APPLICATION NUMBER: 08/472,256 FILING DATE: 07-JUNE-1995 (vii)PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/472,255 FILING DATE: 07-JUNE-1995 (vii)PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/477,829 FILING DATE: 07-JUNE-1995 (viii)ATTORNEY/AGENT INFORMATION: NAME: Barry J. Swanson REGISTRATION NUMBER: 33,215 REFERENCE/DOCKET NUMBER: (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (303) 793-3333 TELEFAX: (303) 793-3433 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGGAAAAGCG AAUCAUACAC AAGANNNNNN NNNNNNNNNN NNNNNNNNNN ECFIED SHEE (RULE 91) WO 96/40703 PCT/S96/09455 125 NNNNNNNNNN NNNNNNNNNN NNNNGCUCCG CCAGAGACCA ACCGAGAA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: UAAUACGACU CACUAUAGGG AAAAGCGAAU CAUACACAAG A 41 ne INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID UUCUCGGUUG GUCUCUGGCG GAGC C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:3: 24 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGGAAAAGCG AAUCAUACAC AAGAAUGGUU GGCCUGGGCG CAGGCUUCGA AGACUCGGCG GGAACGGGAA UGGCUCCGCC AGAGACCAAC CGAGAA 96 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, cytosine (ix) FEATURE: PCT/US96/09455 WO 96/40703 (xi)

GGGAAAAGCG

AAAGUAGUGC

OTHER INFORMATION: All U's are 2'-NH 2 SEQUENCE DESCRIPTION: SEQ ID AAUCAUACAC AAGACAGGCA CUGAAAACUC GGCGGGAACG CGACUCAGAC GCGUGCUCCG CCAGAGACCA ACCGAGAA uracil 98 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 91 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GGGAAAAGCG AAUCAUACAC AAGAAGUCUG GCCAAAGACU CGGCGGGAAC GUAAAACGGC CAGAAUUGCU CCGCCAGAGA CCAACCGAGA A 91 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosii (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGGAAAAGCG AAUCAUACAC AAGAGUAGGA GGUUCCAUCA CCAGGACUCG GCGGGAACGG AAGGUGAUGS GCUCCGCCAG AGACCAACCG AGAA 94 ne INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GGGAAAAGCG AAUCAUACAC AAGAACAAGG AUCGAUGGCG AGCCGGGGAG GGCUCGGCGG GAACGAAAUC UGCUCCGCCA GAGACCAACC GAGAA ne INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCT/US96/09455 127 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AGCG AAUCAUACAC AAGAUUGGGC AGGCAGAGCG AGACCGGGGG GGGA ACGGAACAGG AAUGCUCCGC CAGAGACCAA CCGAGAA 97 ne

GGGAAA

CUCGGC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGAAAGGGA UGGGAUUGGG ACGAGCGGCC AAGACUCGGC GGGAACGAAG GGUGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGAAA GUGUCAUGGU AGCAAGUCCA AUGGUGGACU CUGCUCCGCC AGAGACCAAC CGAGAA 96 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGUGA AGUGGGUAGG UAGCUGAAGA CGGUCUGGGC GCCAGCUCCG CCAGAGACCA ACCGAGAA 98 WO 96/40703 PCT/US96/09455 128 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAAAAGCG

AAGACUCGGC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:13: AAUCAUACAC AAGAAAGGGA UGGGAUUGGG ACGAGCGGCC GGGAACGAAG GGUCCGCUCC GCCAGAGACC AACCGAGAA 99 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAAAAGCG

ACGAUCACUU

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:14: AAUCAUACAC AAGACUCGGC GGGAACGAAG UGUGUGAGUA GGUACUAAAA GCCCGCUCCG CCAGAGACCA ACCGAGAA 98 INFORMAUION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 100 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAAUCGAA AGUGUACUGA AUUAGAACGG UGGGCCUGCU CAUCGUGCUC CGCCAGAGAC CAACCGAGAA 100 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 103 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: PCT/US96/09455 WO 96/40703 (xi)

GGGAAAAGCG

UAGCACGAUG

GAA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:16: AAUCAUACAC AAGACUCGGC GGGAAUCGUA AUGUGGAUGA GCAGYAGUAG UCGGACCGCG CUCCGCCAGA GACCAACCGA 100 103 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGGAAAAGCG AAUCAUACAC AAGACAGCGG CGGAGUCAGU GAAAGCGUGG GGGGYGCGGG AGGUCUACCC UGACGCUCCG CCAGAGACCA ACCGAGAA 98 ne INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GGGAAAAGCG AAUCAUACAC AAGACGGCUG UGUGUGGUAG CGUCAUAGUA GGAGUCGUCA CGAACCAAGG CGCUCCGCCA GAGACCAACC GAGAA ne INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGGAAAAGCG AAUCAUACAC AAGACGGCUG UGUGGUGUUG GAGCGUCAUA GUAGGAGUCG UCACGAACCA AGGCGCUCCG CCAGAGACCA ACCGAGAA 98 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid WO 96/40703 PCT/US96/09455 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID AGCG AAUCAUACAC AAGACGAUGC GAGGCAAGAA AUGGAGUCGU CCCU CUUGCAGUGC GCGGCUCCGC CAGAGACCAA CCGAGAA 97 ne

GGGAAA

UACGAA

INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GGGAAAAGCG AAUCAUACAC AAGACGUGCG GAGCAAAUAG GGGAUCAUGG AGUCGUACGA ACCGUUAUCG CGCUCCGCCA GAGACCAACC GAGAA ne INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GGGAAAAGCG AAUCAUACAC AAGACUGGGG AGCAGGAUAU GAGAUGUGCG GGGCAAUGGA GUCGUGACGA ACCGCUCCGC CAGAGACCAA CCGAGAA 97 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GGGAAAAGCG AAUCAUACAC AAGAGUCCGC CCCCAGGGAU GCAACGGGGU GGCUCUAAAA GGCUUGGCUA AGCUCCGCCA GAGACCAACC GAGAA PCT/US96/09455 WO 96/40703 131 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAAAAGCG

GUGGGUGGCA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:24: AAUCAUACAC AAGAGAGAAU GAGCAUGGCC GGGGCAGGAA ACGGAGGCCA GCUCCGCCAG AGACCAACCG AGAA 94 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: (xi)

GGGAAAAGCG

CCCCUAGGAU

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID AAUCAUACAC AAGAGAUACA GCGCGGGUCU AAAGACCUUG GCAACGGGGU GGCUCCGCCA GAGACCAACC GAGAA INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2 '-NH 2 uracil (xi)

GGGAAAAGCG

CUCGUCCUAG

SEQUENCE DESCRIPTION: SEQ ID NO:26: AAUCAUACAC AAGAUGAAGG GUGGUAAGAG AGAGUCUGAG GGAUGCAACG GCAGCUCCGC CAGAGACCAA CCGAGAA INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 132 (xi)

GGGAAAAGCG

UAGGGUUGCA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:27: AAUCAUACAC AAGACAAACC UGCAGUCGCG CGGUGAAACC ACGGUACAUC GCUGUGCUCC GCCAGAGACC AACCGAGAA 99 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GGGAAAAGCG AAUCAUACAC AAGAGUGGAC UGGAAUCUUC GAGGACAGGA ACGUUCCUAG GGAUGCAACG GACGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGGAAAAGCG AAUCAUACAC AAGAGUGUAC CAAUGGAGGC AAUGCUGCGG GAAUGGAGGC CUAGGGAUGC AACGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGAGUCCCU AGGGAUGCAA CGGGCAGCAU UCGCAUAGGA GUAAUCGGAG GUCGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single RECTIFIED SHEET (RULE 91) WO 96/40703 PCT/US96/09455 133 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGGAAAAGCG AAUCAUACAC AAGAGCCUAG GGAUGCAACG GCGAAUGGAU AGCGAUGUCG UGGACAGCCA GGUGCUCCGC CAGAGACCAA CCGAGAA 97 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AGCG AAUCAUACAC AAGAAUCGAA CCUAGGGAUG CAACGGUGAA GAGG AUUCGCCAUU AGGCGCUCCG CCAGAGACCA ACCGAGAA 98

GGGAAA

GGUUGU

INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GGGAAAAGCG AAUCAUACAC AAGAGCUAGG GAUGCCGCAG AAUGGUCGCG GAUGUAAUAG GUGAAGAUUG UUGCGCUCCG CCAGAGACCA ACCGAGAA 98 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: (xi)

GGGAAAAGCG

UUGAUGCGCG

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:34: AAUCAUACAC AAGAGGACCU AGGGAUGCAA CGGUCCGACC GGUGUCCAAG CUACGCUCCG CCAGAGACCA ACCGAGAA 98 RECTIFIED SHEET (RULE 91) PCT/US96/09455 WO 96/40703 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGAAAGGGA GGAGCUAGAG AGGGAAAGGU UACUACGCGC CAGAAUAGGA UGUGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GGGAAAAGCG AAUCAUACAC AAGACCAACG UACAUCGCGA GCUGGUGGAG AGUUCAUGAG GGUGUUACGG GGUGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GGGAAAAGCG AAUCAUACAC AAGACCCAAC GUGUCAUCGC GAGCUGGCGG AGAGUUCAUG AGGGUUACGG GUGCUCCGCC AGAGACCAAC CGAGAA 96 ne INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: PCT/US96/09455 WO 96/40703 135 (xi)

GGGAAAAGCG

GGUAGGCGGU

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:38: AAUCAUACAC AAGAGUUGGU GCGAGCUGGG GCGGCGAGAA CCGAGUGUUC GAAUGCUCCG CCAGAGACCA ACCGAGAA 98 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: AGCG AAUCAUACAC AAGACUGGCA AGRAGUGCGU GAGGGUACGU UGUU UGGGCCGAUC GCAUGCUCCG CCAGAGACCA ACCGAGAA 2 cytosine uracil 98

GGGAAA

UAGGGG

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: (xi)

GGGAAAAGCG

AGAGGGAUUG

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID AAUCAUACAC AAGAUUGGUC GUACUGGACA GAGCCGUGGU GGACAAAGUG UCAGCUCCGC CAGAGACCAA CCGAGAA 97 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: (xi)

GGGAAAAGCG

GUCGGUGGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:41: AAUCAUACAC AAGAUGUGAG AAAGUGGCCA ACUUUAGGAC UGYGCGGGUA GGCUCGCUCC GCCAGAGACC AACCGAGAA 99 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCT/US96/09455 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GGGAAAAGCG AAUCAUACAC AAGACAGGCA GAUGUGUCUG AGUUCGUCGG AGUAGACGUC GGUGGACGCG GAACGCUCCG CCAGAGACCA ACCGAGAA 98 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GGGAAAAGCG AAUCAUACAC AAGAUGUGAU UAGGCAGUUG CAGCCGCCGU GCGGAGACGU GACUCGAGGA UUCGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GGGAAAAGCG AAUCAUACAC AAGAUGCCGG UGGAAAGGCG GGUAGGUGAC CCGAGGAUUC CUACCAAGCC AUGCUCCGCC AGAGACCAAC CGAGAA 96 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 93 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGAGAGGUG RAUGGGAGAG UGGAGCCCGG GUGACUCGAG GAUUCCCGUG CUCCGCCAGA GACCAACCGA GAA 93 ne PCTIUS96/09455 WO 96/40703 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGGAAAAGCG AAUCAUACAC AAGAGUCAUG CUGUGGCUGA ACAUACUGGU GAAAGUUCAG UAGGGUGGAU ACAGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGGAAAAGCG AAUCAUACAC AAGACCGGGG AUGGUGAGUC GGGCAGUGUG ACCGAACUGG UGCCCGCUGA GAGCUCCGCC AGAGACCAAC CGAGAA 96 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GGGAAAAGCG AAUCAUACAC AAGAACACUA ACCAGGUCUC UGAACGCGGG ACGGAGGUGU GGGCGAGGUG GAAGCUCCGC CAGAGACCAA CCGAGAA 97 ne INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: PCT/US96/09455 WO 96/40703 (xi)

GGGAAAAGCG

CGUGCUGAAG

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:49: AAUCAUACAC AAGACCGUCU CCCGAGAACC AGGCAGAGGA GAGCUGCAUC UAGAAGCUCC GCCAGAGACC AACCGAGAA 99 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGACCGUCU CCGAGAACCA GGCAGAGGAG GUGCUGAAGG RGCUGGCAUC UACAAGCUCC GCCAGAGACC AACCGAGAA 99 ne INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2 '-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: GGGAAAAGCG AAUCAUACAC AAGACCCGCA CAUAAUGUAG GGAACAAUGU UAUGGCGGAA UUGAUAACCG GUGCUCCGCC AGAGACCAAC CGAGAA 96 ne INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GGGAAAAGCG AAUCAUACAC AAGACGAUGU UAGCGCCUCC GGGAGAGGUU AGGGUCGUGC GGNAAGAGUG AGGUGCUCCG CCAAGAGCCA ACCGAGAA 98 ne INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 99 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCT/US96/09455 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GGGAAAAGCG AAUCAUACAC AAGAGGUACG GGCGAGACGA GAUGGACUUA UAGGUCGAUG AACGGGUAGC AGCUCGCUCC GCCAGAGACC AACCGAGAA 99 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GGGAAAAGCG AAUCAUACAC AAGACGGUUG CUGAACAGAA CGUGAGUCUU GGUGAGUCGC ACAGAUUGUC CUGCUCCGCC AGAGACCAAC CGAGAA 96 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosiz (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAAAAGCG AAUCAUACAC AAGAACUGAG UAAGGUCUGG CGUGGCAUUA GGUUAGUGGG AGGCUUGGAG UAGGCUCCGC CAGAGACCAA CCGAGAA 97 ie INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID AAGACUCGGC GGGAACGAAA INFORMATION FOR SEQ ID NO:57: C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:56: PCT/US96/09455 WO 96/40703 140 SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID GGAGUCGUGA CGAACC INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID CCUAGGGAUG CAACGG C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:57: 16 C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:58: 16 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID RCUGGGAGRG UGGGUGUU C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:59: 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID UGUGNNNNAG UNNNNNNNNN UAGACGUCGG UGGACNNNGC GG 42 ne WO 96/40703 PCT/US96/09455 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID GGGNNNGUGA CYCGRGGAYU C C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO: 61: 21 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID UGANCNNACU GGUGNNNGNG NAG C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:62: 23 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID GUCUCYGAAC NNGGNAGGAN GUGNUGGAGN UG C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:63: 32 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN ie PCT/US96/09455 WO 96/40703 NNNNNCAGAC GACUCGCCCG A INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID TAATACGACT CACTATAGGG AGGACGATGC GG INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID TCGGGCGAGT CGTCCTG C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil 32 C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:66: 17 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: GGGAGGACGA UGCGGCGCGU AUGUGUGAAA GCGUGUGCAC GGAGGCGUCU ACAAUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: PCTfUS96/09455 WO 96/40703 (xi)

GGGAGGACGA

CAGCACAGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:68: UGCGGGGCAU UGUGUGAAUA GCUGAUCCCA CAGGUAACAA GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: GGGAGGACGA UGCGGUAAUG UGUGAAUCAA GCAGUCUGAA UAGAUUAGAC AAAAUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAGGACGA UGCGGAUGUG UGAGUAGCUG AGCGCCCGAG UAUGAWACCU GACUACAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: GGGAGGACGA UGCGGAAACC UUGAUGUGUG AUAGAGCAUC CCCCAGGCGA CGUACCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single PCT/US96/09455 WO 96/40703 144 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GGGAGGACGA UGCGGUUGAG AUGUGUGAGU ACAAGCUCAA AAUCCCGUUG GAGGCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GGGAGGACGA UGCGGUAGAG GUAGUAUGUG UGGGAGAUGA AAAUACUGUG GAAAGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: GGGAGGACGA UGCGGAAAGU UAUGAGUCCG UAUAUCAAGG UCGACAUGUG UGAAUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAGGACGA UGCGGCACGA AAAACCCGAA UUGGGUCGCC CAUAAGGAUG UGUGACAGAC GACUCGCCCG A 71 ne WO 96/40703 PCT/US96/09455 145 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GGGAGGACGA UGCGGGUAAA GAGAUCCUAA UGGCUCGCUA GAUGUGAUGU GAAACCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: GGGAGGACGA UGCGGUAACA ACAAUCAAGG CGGGUUCACC GCCCCAGUAU GAGUGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GGGAGGACGA UGCGGUAACA ACAAUCAAGG CGGGUUYACC GCCCCAGUAU GAGUACAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 (xi)

GGGAGGACGA

GAGUACAGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:79: UGCGGUAACA ACAAUCAAGG CGGGUUYACC GCUCCAGUAU GACUCGCCCG A 71 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAGGACGA UGCGGUAACA ACAAUCAAGG CGGGUUCACC GCCCCAGUAU GAGUGCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: GGGAGGACGA UGCGGACCAA GCAAUCUAUG GUCGAACGCU ACACAUGAAU GACGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: ACGA UGCGGGAACA UGAAGUAAUC AAAGUCGUAC CAAUAUACAG

GGGAGG

GAAGCCAGAC GACUCGCCCG A INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single PCTIUS96/09455 WO 96/40703 147 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi)

GGGAGGACGA

AAUACAGACG

SEQUENCE DESCRIPTION: SEQ ID NO:83: UGCGGGACAU GAAGUAAGAC CGUCACAAUU CGAAUGAUUG

ACUCGCCCGA

INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAGGACGA

CCAAAACAGA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:84: UGCGGGAACA UGAAGUAAAA AGUCGACGAA UUAGCUGUAA CGACUCGCCC GA 72 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID rACGA UGCGGGAACA UGAAGUAAAA GUCUGAGUUA GUAAAUUACA !AGAC GACUCGCCCG A cytosine uracil 71

GGGAGG

GUGAUC

INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: GGGAGGACGA UGCGGGAACU UGAAGUUGAA NUCGCUAAGG UUAUGGAUUC AAGAUUCAGA CGACUCGCCC GA 72 WO 96/40703 PCTIS96/09455 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: GGGAGGACGA UGCGGAACAU GAAGUAAUAA GUCGACGUAA UUAGCUGUAA CUAAACAGAC GACUCGCCCG A cytosine Suracil 71 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: GGGAGGACGA UGCGGAACAU GAAGUAAAAG UCUGAGUUAG AAAUUACAAG UGAUCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: GGGAGGACGA UGCGGUAACA UAAAGUAGCG CGUCUGUGAG AGGAAGUGCC UGGAUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 149 (xi)

GGGAGGACGA

UGAGACAGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID UGCGGAUAGA ACCGCAAGGA UAACCUCGAC CGUGGUCAAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: GGGAGGACGA UGCGGUAAGA ACCGCUAGCG CACGAUCAAA CAAAGAGAAA CAAACAGACG ACUCGCCCGA ne INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: GGGAGGACGA UGCGGUUCUC UCCAAGAACY GAGCGAAUAA ACSACCGGAS UCACACAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: GGGAGGACGA UGCGGUGUCU CUCCUGACUU UUAUUCUUAG UUCGAGCUGU CCUGGCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCT/US96/09455 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi)

GGGAGGACGA

AUCCCCAGAC

SEQUENCE DESCRIPTION: SEQ ID NO:94: UGCGGCCGUA CAUGGUAARC CUCGAAGGAU UCCCGGGAUG GACUCGCCCG A INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2 '-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID ACGA UGCGGUCCCA GAGUCCCGUG AUGCGAAGAA UCCAUUAGUA AGAC GACUCGCCCG A cytosine uracil 71

GGGAGG

CCAGAC

INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: ACGA UGCGGGAUGU AAAUGACAAA UGAACCUCGA AAGAUUGCAC GACG ACUCGCCCGA ne

GGGAGG

ACUCCA

INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: GGGAGGACGA UGCGGAUGUA AAUCUAGGCA GAAACGUAGG GCAUCCACCG CAACGACAGA CGACUCGCCC GA 72 WO 96/40703 PCTIUS96/09455 151 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: GGGAGGACGA UGCGGAUAAC CCAAGCAGCN UCGAGAAAGA GCUCCAUAGA UGAUCAGACG ACUCGCCCGA ne INFORMATION FOR SEQ ID N0:99: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: GGGAGGACGA UGCGGCAAAG CACGCGUAUG GCAUGAAACU GGCANCCCAA GUAAGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: GGGAGGACGA UGCGGCAAAA GGUUGACGUA GCGAAGCUCU CAAAAUGGUC AUGACCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 (xi)

GGGAGGACGA

ACCACAGACG

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:101: UGCGGAAGUG AAGCUAAAGC GGAGGGCCAU UCAGUUUCNC ACUCGCCCGA INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: GGGAGGACGA UGCGGAAGUG AAGCUAAAGS GGAGGGCCAC UCAGAAACGC ACCACAGACG ACUCGCCCGA ne INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: GGGAGGACGA UGCGGCACCG CUAAGCAGUG GCAUAGCCCA GUAACCUGUA AGAGACAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 67 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: GGGAGGACGA UGCGGCACGC UAAGCAGUGG CAUAGCGWAA CCUGUAAGAG ACAGACGACU CGCCCGA 67 INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single PCTIUS96/09455 WO 96/40703 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: GGGAGGACGA UGCGGAGAUU ACCAUAACCG CGUAGUCGAA GACAUAUAGU AGCGACAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: GGGAGGACGA UGCGGACUCG GGUAGAACGC GACUUGCCAC CACUCCCAUA AAGACCAGAC GACUCGCCCG A 71 ie INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: GGGAGGACGA UGCGGUCAGA ACUCUGCCGC UGUAGACAAA GAGGAGCUUA GCGAACAGAC GACUCGCCCG A cytosine uracil 71 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: GGGAGGACGA UGCGGAAUGA GCAUCGAGAG AGCGCGAACU CAUCGAGCGU ACUAACAGAC GACUCGCCCG A 71 ie PCT/US96/09455 WO 96/40703 INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: GGGAGGACGA UGCGGCAAAG CACGCGUAUG GCAUGAAACU GGCANCCCAA GUAAGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: GGGAGGACGA UGCGGGAUGC AGCAACCUGA AAACGGCGUC CACAGGUAAU AACAGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:ll1: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: GGGAGGACGA UGCGGAAACU CGCUACAAAC ACCCAAUCCU AGAACGUUAU GGAGACAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:112: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: PCT/US96/09455 WO 96/40703 (xi)

GGGAGGACGA

GAUCACAGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:112: UGCGGCUAGC AUAGCCACCG GAACAGACAG AUACGAGCAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAGGACGA

AUAGGCAGAC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:113: UGCGGGAUUC GGAGUACUGA AAAACAACCC UCAAAAGUGC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: GGGAGGACGA UGCGGGUCCA GGACGGACCG CAGCUGUGAU ACAAUCGACU UACACCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: GGGAGGACGA UGCGGAAACU CGCUACAAAC ACCCAAUCCU AGAACGUUAU GGAGACAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single PCT/US96/09455 WO 96/40703 TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: GGGAGGACGA UGCGGCGGCC CUUAUCGGAG GUCUGCGCCA CUAAUUACAU CCACCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH: 67 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: GGGAGGACGA UGCGGUCCAG AGCGUGAAGA UCAACGUCCC GGNGUCGAAG ACAGACGACU CGCCCGA 67 INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 8 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are (ix) FEATURE: OTHER INFORMATION: All U's are (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: 2'-NH 2 cytosine 2'-NH 2 uracil

AUGUGUGA

INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: 8 C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:119: (xi)

CAACAAUCAU

OTHER INFORMATION: All SEQUENCE DESCRIPTION: SEQ ID

GAGUR

INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT[US96/09455 LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID AACAUGAAGU AAGUCARUUA G C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:120: 21 INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 11 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:121: 11 AGAACCGCWA G INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 7 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:122: 7

UCUCUCC

INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 10 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID

CGAAGAAUYC

INFORMATION FOR SEQ ID NO:124: C's are 2'-NH 2 cytosine U's are 2'-NH 2 uracil NO:123: PCT/US96/09455 WO 96/40703 SEQUENCE CHARACTERISTICS: LENGTH: 8 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: AUGUAAAU 8 INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 8 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: AACCCAAG 8 INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: CTACCTACGA TCTGACTAGC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNN GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127: CTACCTACGA TCTGACTAGC INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA PCTIUS96/09455 WO 96/40703 (xi) FEATURE: OTHER INFORMATION: N AT POSITION 2 AND 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: ANANAGGAAC TACATGAGAG TAAGC INFORMATION FOR SEQ ID NO:129: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: CTACCTACGA TCTGACTAGC GGAACACGTG AGGTTTACAA GGCACTCGAC GTAAACACTT GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:130: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130: CTACCTACGA TCTGACTAGC CCCCGAAGAA CATTTTACAA GGTGCTAAAC GTAAAATCAG GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:131: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: CTACCTACGA TCTGACTAGC GGCATCCCTG AGTCATTACA AGGTTCTTAA CGTAATGTAC GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:132: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: CTACCTACGA TCTGACTAGC TGCACACCTG AGGGTTACAA GGCGCTAGAC GTAACCTCTC GCTTACTCTC ATGTAGTTCC IS BIOTIN INFORMATION FOR SEQ ID NO:133: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US96/09455 WO 96/40703 160 (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: CTACCTACGA TCTGACTAGC CACGTTTCAA GGGGTTACAC GAAACGATTC ACTCCTTGGC GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:134: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: CTACCTACGA TCTGACTAGC CGGACATGAG CGTTACAAGG TGCTAAACGT AACGTACTTG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:135: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: CTACCTACGA TCTGACTAGC CGCATCCACA TAGTTCAAGG GGCTACACGA AATATTGCAG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:136: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: CTACCTACGA TCTGACTAGC TACCCCTTGG GCCTCATAGA CAAGGTCTTA AACGTTAGCG CTTACTCTCA TGTAGTTCC 79 79 79 79 INFORMATION FOR SEQ ID NO:137: SEQUENCE CHARACTERISTICS: (ii) (xi)

CTACCTACGA

GTAACGTTGG

LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:137: TCTGACTAGC CACATGCCTG ACGCGGTACA AGGCCTGGAC CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:138: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCTIUS96/09455 WO 96/40703 (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: CTACCTACGA TCTGACTAGC TAGTGCTCCA CGTATTCAAG GTGCTAAACG AAGACGGCCT GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:139: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: CTACCTACGA TCTGACTAGC AGCGATGCAA GGGGCTACAC GCAACGATTT AGATGCTCTG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:140: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: CTACCTACGA TCTGACTAGC CCAGGAGCAC AGTACAAGGT GTTAAACGTA ATGTCTGGTG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:141: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

GTAACGTGTG

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:141: TCTGACTAGC ACCACACCTG GGCGGTACAA GGAGTTATCC CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:142: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:142: TCTGACTAGC CAAGGTAACC AGTACAAGGT GCTAAACGTA CTTACTCTCA TGTAGTTCC (ii) (xi)

CTACCTACGA

ATGGCTTCGG

INFORMATION FOR SEQ ID NO:143: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/40703 PCT/US96/09455 (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143: CTACCTACGA TCTGACTAGC ACCCCCGACC CGAGTACAAG GCATTCGACG TAATCTGGTG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:144: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144: CTACCTACGA TCTGACTAGC CAGTACAAGG TGTTAAACGT AATGCCGATC GAGTTGTATG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:145: SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145: CTACCTACGA TCTGACTAGC ACAACGAGTA CAAGGAGATA GACGTAATCG GCGCAGGTAT CGCTTACTCT CATGTAGTTC C INFORMATION FOR SEQ ID NO:146: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

CCGACCACGG

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:146: TCTGACTAGC CACGACAGAG AACAAGGCGT TAGACGTTAT CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:147: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single (ii) (xi)

CTACCTACGA

CACTTCACCT

TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:147: TCTGACTAGC AGGGAGAACA AGGTGCTAAA CGTTTATCTA GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:148: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US96/09455 WO 96/40703 (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148: CTACCTACGA TCTGACTAGC AGGACCAAGG TGTTAAACGG CTCCCCTGGC TATGCCTCTT GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:149: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149: CTACCTACGA TCTGACTAGC TACACAAGGT GCTAAACGTA GAGCCAGATC GGATCTGAGC GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:150: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150: CTACCTACGA TCTGACTAGC GGACAAGGCA CTCGACGTAG TTTATAACTC CCTCCGGGCC GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:151: SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151: CTACCTACGA TCTGACTAGC TACACAAGGG GCCAAACGGA GAGCCAGACG CGGATCTGAC AGCTTACTCT CATGTAGTTC C INFORMATION FOR SEQ ID NO:152: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152: CTACCTACGA TCTGACTAGC CGGCTATACN NGGTGCTAAA CGCAGAGACT CGATCAACAG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:153: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCTILS96/09455 WO 96/40703 164 (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153: CTACCTACGA TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA TGGAAGCTTG GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:154: SEQUENCE CHARACTERISTICS: LENGTH: 73 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

TGGGCTTACT

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:154: TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA CTCATGTAGT TCC INFORMATION FOR SEQ ID NO:155: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155: ACGA TCTGACTAGC GAGTAGCCAA GGCGTTAGAC GGAGGGGGAA CACA GCTTACTCTC ATGTAGTTCC

CTACCT

TGTGAG

INFORMATION FOR SEQ ID NO:156: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156: 'ACGA TCTGACTAGC TAGCTCCACA CACAASSCGC RGCACATAGG CTGG GCTTACTCTC ATGTAGTTCC

CTACCT

GGATAT

INFORMATION FOR SEQ ID NO:157: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:157: TCTGACTAGC CATCAAGGAC TTTGCCCGAA ACCCTAGGTT GCTTACTCTC ATGTAGTTCC (ii) (xi)

CTACCTACGA

CACGTGTGGG

INFORMATION FOR SEQ ID NO:158: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCTIUS96/09455 WO 96/40703 (ii) (xi)

CTACCTACGA

CTGCGGGTGG

MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:158: TCTGACTAGC CATTCACCAT GGCCCCTTCC TACGTATGTT CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:159: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single (ii) (xi)

CTACCTACGA

GTTCCATCCG

TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:159: TCTGACTAGC GCAACGTGGC CCCGTTTAGC TCATTTGACC GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:160: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) (xi)

CTACCTACGA

TGGGTGTCTG

MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:160: TCTGACTAGC CCACAGACAA TCGCAGTCCC CGTGTAGCTC CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:161: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:161: TCTGACTAGC CCACCGTGAT GCACGATACA TGAGGGTGTG CTTACTCTCA TGTAGTTCC (ii) (xi)

CTACCTACGA

TCAGCGCATG

INFORMATION FOR SEQ ID NO:162: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

CAAARCRGTR

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:162: TCTGACTAGC CGAGGTAGTC GTTATAGGGT RCRCACGACA GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:163: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCTIUS96/09455 WO 96/40703 (ii) (xi)

CTACCTACGA

GGGAAGTGAG

MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:163: TCTGACTAGC TGGCGGTACG GGCCGTGCAC CCACTTACCT CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:164: SEQUENCE CHARACTERISTICS: (ii) (xi)

CTACCTACGA

GCGTAATCAT

LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:164: TCTGACTAGC CTCTGCTTAC CTCATGTAGT TCCAAGCTTG GGCTTACTCT CATGTAGTTC C INFORMATION FOR SEQ ID NO:165: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:165: TCTGACTAGC AGCGTTGTAC GGGGTTACAC ACAACGATTT CTTACTCTCA TGTAGTTCC (ii) (xi)

CTACCTACGA

AGATGCTCTG

INFORMATION FOR SEQ ID NO:166: SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

CTCAGAGGAC

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:166: TCTGACTAGC TGATGCGACT TTAGTCGAAC GTTACTGGGG AGCTTACTCT CATGTAGTTC C INFORMATION FOR SEQ ID NO:167: SEQUENCE CHARACTERISTICS: LENGTH: 81 base pairs TYPE: nucleic acid STRANDEDNESS: single (ii) (xi)

CTACCTACGA

CACNCAGGCT

TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:167: TCTGACTAGC CGAGGATCTG ATACTTATTG AACATAMCCG TGCTTACTCT CATGTAGTTC C INFORMATION FOR SEQ ID NO:168: SEQUENCE CHARACTERISTICS: LENGTH: 73 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US96/09455 WO 96/40703 (ii) MOLECULAR TYPE:DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168: CTACCTACGA TCTGACTAGC CGATCGTGTG TCATGCTACC TACGATCTGA CTAGCTTACT CTCATGTAGT TCC INFORMATION FOR SEQ ID NO:169: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169: CTACCTACGA TCTGACTAGC GCACACAAGT CAAGCATGCG ACCTTCAACC ATCGACCCGA GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:170: (i) SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid (ii) (xi)

CTACCTACGA

GACANNNNNN

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:170: TCTGACTAGC ATGCCAGTGC AGGCTTCCAT CCATCAGTCT GCTTACTCT CATGTAGTTCC 73 79 INFORMATION FOR SEQ ID NO:171: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171: CTACCTACGA TCTGACTAGC CACTTCGGCT CTACTCCACC TCGGTCCTCC ACTCCACAG GCTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:172: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs (ii) (xi)

CTACCTACGA

TCCTCATCGC

TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:172: TCTGACTAGC CGCTAACTGA CCCTCGATCC CCCCAAGCCA GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:173: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US96/09455 WO 96/40703 (ii) (xi)

CTACCTACGA

ATGGCTTCGG

MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:173: TCTGACTAGC ATCTGACTAG CTCGGCGAGA GTACCCGCTC CGAATGCCCT GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:174: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174: CTACCTACGA TCTGACTAGC TCCTGAGACG TTACAATAGG CTGCGGTACT GCAACGTGGA GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:175: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175: CTACCTACGA TCTGACTAGC CGGCAGGGCA CTAACAAGGT GTTAAACGTT ACGGATGCCG CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:176: SEQUENCE CHARACTERISTICS: LENGTH: 90 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176: CTACCTACGA TCTGACTAGC TGCACACCGG CCCACCCGGA CAAGGCGCTA GACGAAATGA CTCTGTTCTG GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:177: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177: CTACCTACGA TCTGACTAGC GACGAAGAGG CCAAGGTGAT AACCGGAGTT TCCGTCCGCG CTTACTCTCA TGTAGTTCC 79 79 INFORMATION FOR SEQ ID NO:178: SEQUENCE CHARACTERISTICS: LENGTH: 79 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear PCT/US96/09455 WO 96/40703 (ii) (xi)

CTACCTACGA

AGAGCCCGAG

MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:178: TCTGACTAGC AAGGACTTAG CTATCCAAGG CACTCGACGA CTTACTCTCA TGTAGTTCC INFORMATION FOR SEQ ID NO:179: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179: CTACCTACGA TCTGACTAGC ATGCCCAGTT CAAGGTTCTG ACCGAAATGA CTCTGTTCTG GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:180: SEQUENCE CHARACTERISTICS: LENGTH: 80 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180: CTACCTACGA TCTGACTAGC GCAGCGTGGC CCTGTTTAGC TCATTTGACC GTTCCATCCG GCTTACTCTC ATGTAGTTCC INFORMATION FOR SEQ ID NO:181: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181: TACAAGGYGY TAVACGTA INFORMATION FOR SEQ ID NO:182: SEQUENCE CHARACTERISTICS: LENGTH: 8 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:

GGCCCCGT

INFORMATION FOR SEQ ID NO:183: SEQUENCE CHARACTERISTICS: LENGTH: 10 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183: PCT/US96/09455 WO 96/40703

RCACGAYACA

INFORMATION FOR SEQ ID NO:184: SEQUENCE CHARACTERISTICS: LENGTH: 7 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:

CTTACCT

INFORMATION FOR SEQ ID NO:185: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185: TAGCCAAGGT AACCAGTACA AGGTGCTAAA CGTAATGGCT TCGGCTTAC INFORMATION FOR SEQ ID NO:186: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186: GTAACCAGTA CAAGGTGCTA AACGTAATGG CTTCGGCTTA C INFORMATION FOR SEQ ID NO:187: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187: CCAGTACAAG GTGCTAAACG TAATGG INFORMATION FOR SEQ ID NO:188: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188: CGCGGTAACC AGTACAAGGT GCTAAACGTA ATGGCGCG 41 26 INFORMATION FOR SEQ ID NO:189: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs WO 96/40703 PCT/US96/09455 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:189: GTACAAGGTG CTAAACGTAA TGGCGC (ii) (xi)

GCGGTAACCA

INFORMATION FOR SEQ ID NO:190: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190: ACATGAGCGT TACAAGGTGC TAAACGTAAC GTACTTGCTT ACTCTCATGT INFORMATION FOR SEQ ID NO:191: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191: CGCGCGTTAC AAGGTGCTAA ACGTAACGTA CTTGCTTACT CGCG 44 INFORMATION FOR SEQ ID NO:192: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192: GCGTTACAAG GTGCTAAACG TAACGT 26 INFORMATION FOR SEQ ID NO:193: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: (ix) FEATURE: OTHER INFORMATION: N at position 1 is an amino modifier C6 dT (ix) FEATURE: OTHER INFORMATION: Nucleotide 51 is an invertedorientation linkage) phosphoramidite (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193: NTAGCCAAGG TAACCAGTAC AAGGTGCTAA ACGTAATGGC TTCGGCTTAC TT 52 INFORMATION FOR SEQ ID NO:194: WO 96/40703 PCT/US96/09455 172 SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194: TAGCCATTCA CCATGGCCCC TTCCTACGTA TGTTCTGCGG GTGGCTTA INFORMATION FOR SEQ ID NO:195: SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195: AGCTGGCGGT ACGGGCCGTG CACCCACTTA CCTGGGAAGT GAGCTTA INFORMATION FOR SEQ ID NO:196: SEQUENCE CHARACTERISTICS: .LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (ix) FEATURE: OTHER INFORMATION: N modifier C6 dT at position 1 is an amimo (ix) FEATURE: OTHER INFORMATION: Nucleotide number 28 is an inverted-orientation linkage) phosphoramidite (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196: NCCAGTACAA GGTGCTAAAC GTAATGGTT 29 INFORMATION FOR SEQ ID NO:197: SEQUENCE CHARACTERISTICS: LENGTH:40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197: TAATACGACT CACTATAGGG AGACAAGAAT AAACGCTCAA INFORMATION FOR SEQ ID NO:198: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198: GCCTGTTGTG AGCCTCCTGT CGAA 24 WO 96/40703 PCT/US96/09455 INFORMATION FOR SEQ ID NO:199: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199: GGGAGACAAG AAUAAACGCU CAACGAAUCA GUAAACAUAA CACCAUGAAA CAUAAAUAGC ACGCGAGACG UCUUCGACAG GAGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:200: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200: GGGAGACAAG AAUAAACGCU CAACGAGUUC ACAUGGGAGC AAUCUCCGAA UAAACAACAC GCKAKCGCAA AUUCGACAGG AGGCUCACAA CAGGC ne INFORMATION FOR SEQ ID NO:201: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201: GGGAGACAAG AAUAAACGCU CAACGACCAC AAUACAAACU CGUAUGGAAC ACGCGAGCGA CAGUGACGCA UUUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:202: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 (xi)

GGGAGACAAG

GAAAACAAAU

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:202: AAUAAACGCU CAACGUCAAG CCAGAAUCCG GAACACGCGA CAACGACCAA UCGAUUCGAC AGGAGGCUCA CAAAGGC 97 INFORMATION FOR SEQ ID NO:203: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203: GGGAGACAAG AAUAAACNCU CAACGACCAC AAUAACCGGA AAUCCCCGCG GUUACGGAAC ACGCGAACAU GAAUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:204: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204: GGGAGACAAG AAUAAACGCU CAACGAACCA CGGGGAAAUC CACCAGUAAC ACGCGAGGCA AACAGACCCU CUUCGACAGG AGGCUCACAA CAGGC ne INFORMATION FOR SEQ ID NO:205: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205: GGGAGACAAG AAUAAACGCU CAACGAGCAA AAGUACUCAC GGGACCAGGA GAUCAGCAAC ACGCGAGACG AAAUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:206: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCT/US96/09455 TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206: LCAAG AAUAAACGCU CAACGAGCCA GGAACAUCGA CGUCAGCAAA ICGCA ACCAGUAACA CCUUCGACAG GAGGCUCACA ACAGGC 96 ne

GGGAGA

CGCGAG

INFORMATION FOR SEQ ID NO:207: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:207: GGGAGACAAG AAUAAACGCU CAACGCACCA GGAACAACGA GAACCAUCAG UAAACGCGAG CGAUUGCAUG UUCGACAGGA GGCUCACAAC AGGC 94 INFORMATION FOR SEQ ID NO:208: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208: GGGAGACAAG AAUAAACGCU CAACGCACCA GGAACAACAA GAACCAUCAG UAAGCGCGAG CGAUUGCAUA UUCGACAGGA GGCUCACAAC AGGC 94 INFORMATION FOR SEQ ID NO:209: SEQUENCE CHARACTERISTICS: LENGTH: 101 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209: GGGAGACAAG AAUAAACGCU CAACGAGCAA GGAACGAAUA CAAACCAGGA AACUCAGCAA CACGCGAGCA GUAAGAAUUC GACAGGAGGC UCACAACAGG 100 C 101 WO 96/40703 PCTIUS96/09455 INFORMATION FOR SEQ ID NO:210: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAGACAAG

GAUCAGCAUU

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:210: AAUAAACGCU CAACAGUUCA CUCAACCGGC ACCAGACUAC GGCGAGUGAA CACUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:211: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211: GGGAGACAAG AAUAAACGCU CAACUGGCAA CGGGAUAACA ACAAAUGUCA CCAGCACUAG CGAGACGGAA GGUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:212: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212: GGGAGACAAG AAUAAACGCU CAACGAUGAG CGUGACCGAA GCUAUAAUCA GGUCGAUUCA CCAAGCAAUC UUAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:213: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: WO 96/40703 PCT/US96/09455 (xi)

GGGAGACAAG

UAAGUAUUGA

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:213: AAUAAACGCU CAAAGGAUCA CACAAACAUC GGUCAAUAAA UAGCGGGGAU AUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:214: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214: GGGAGACAAG AAUAAACGCU CAACAACCCA ACCAUCUAGA GCUUCGAACC AUGGUAUACA AGGGAACACA AAAUUCGCGG AGGCUCCAAC AGGCGGC 97 ne INFORMATION FOR SEQ ID NO:215: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY:linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215: GGGAGACAAG AAUAAACGCU CAAGCGGUCA GAAACAAUAG CUGGAUACAU ACCGCGCAUC CGCUGGGCGA UAUUCGACAG GAGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:216: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216: GGGAGACAAG AAUAAACGCU CAAACAAGAG AGUCAAACCA AGUGAGAUCA GAGCGUUUAG CGCGGAAAGC ACAUUCGACA GGAGGCUCAC AACAGGC 97 ne ne INFORMATION FOR SEQ ID NO:217: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single WO 96/40703 PCTfUS96/09455 178 TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217: GGGAGACAAG AAUAAACGCU CAAACUCGAC UAGUAAUCAC CCUAGCAUAA AUCUCCUCGA GCACAGACGA UAUUCGACAG GAGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:218: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218: GGGAGACAAG AAUAAACGCU CAAUCAGCAG UAAGCGAUCC UAUAAAGAUC AACUAGCCAA AGAUGACUUA UUCGACAGGA GGCUCACAAC AGGC cytosine uracil 94 INFORMATION FOR SEQ ID NO:219: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAGACAAG

AGACUUCAAG

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:219: AAUAAACGCU CAAAAAGACG UAUUCGAUUC GAAACGAGAA UGAGCCCGCA GUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:220: SEQUENCE CHARACTERISTICS: LENGTH: 49 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220: GAAU CAGUAAACAU AACACCAUGA AACAUAAAUA GCACGCGAG cytosine uracil 49

CUCAAC

INFORMATION FOR SEQ ID NO:221: WO 96/40703 PCT/US96/09455 SEQUENCE CHARACTERISTICS: LENGTH: 47 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221: 'GAGU UCACAUGGGA GCAAUCUCCG AAUAAACAAC ACGCGAG 47 ne

CUCAAC

INFORMATION FOR SEQ ID N0:222: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222: CUCAACGAAC CACGGGGAAA UCCACCAGUA ACACGCGAG 39 INFORMATION FOR SEQ ID N0:223: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223: CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:224: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224: CGCUCAACGA GCCAGGAACA UCGACGUCAG CAAACGCGAG CG 42 PCT/US96/09455 WO 96/40703 INFORMATION FOR SEQ ID NO:225: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID CUCAACGAGC CAGGACUACG AUCAGCAAAC GCGAG C's are 2'-F cytosine U's are 2'-F uracil NO:225: INFORMATION FOR SEQ ID NO:226: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226: CUCAACGCAC CAGGAACAAC GAGAACCAUC AGUAAACGCG AG 4 ne INFORMATION FOR SEQ ID NO:227: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227: CUCAACGCAC CAGGAACAAC AAGAACCAUC AGUAAGCGCG AG 42 INFORMATION FOR SEQ ID NO:228: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228: CACUCAACCG GCACCAGACU ACGAUCAGCA UUGGCGAGUG 4 ne PCT/US96/09455 WO 96/40703 INFORMATION FOR SEQ ID NO:229: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229: GAAUCCGGAA CACGCGAGAA AACAAAUCAA CGACCAAUCG AUUCG INFORMATION FOR SEQ ID NO:230: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION: 7, 9, 14, 21 OTHER INFORMATION: G are 2'-O-methyl guanine (ix) FEATURE: LOCATION: 8, 15, 18, 22, 27, 31 OTHER INFORMATION: A are 2'-0-methly adenine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230: CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:231: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION:7, 9, 13, 14, 21, 24, 28 OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE: LOCATION:8, 15, 18, 22, 27, 30, 31 OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231: CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:232: WO 96/40703 PCT/US96/09455 182 SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION: 7, 9, 14, 21, 36 OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE: LOCATION:8, 15, 18, 22, 27, 31, 37 OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232: CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:233: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION: 7, 9, 13, 14, 21, 24, 28, 36 OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE: LOCATION: 8, 15, 18, 22, 27, 30, 31, 37 OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233: CUCAACGAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:234: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION:7, 9, 14 OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE: LOCATION:8, 15, 18, 27, 31 WO 96/40703 PCTIUS96/09455 (xi)

CUCAACGAGC

OTHER INFORMATION: A are 2'-O-methyl-adenine SEQUENCE DESCRIPTION: SEQ ID NO:234: CAGGAACAUC GACGUCAGCA AACGCGAG 38 INFORMATION FOR SEQ ID NO:235: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE: LOCATION: 7, 9, 13, 14, 24 OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE: LOCATION: 8, 15, 18, 22, 27, 31 OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235: GAGC CAGGAACAUC GACGUCAGCA AACGCGAG 38

CUCAAC

INFORMATION FOR SEQ ID NO:236: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236: CUCAACGAGC AAAAGUACUC ACGGGACCAG GAGAUCAGCA ACACGCGAGA CGAAAUUCG 59 INFORMATION FOR SEQ ID NO:237: SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:237: ACGA CCACAAUACA AACUCGUAUG GAACACGCGA GCG 43

CGCUCA

INFORMATION FOR SEQ ID NO:238: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238: ACUG GCAACGGGAU AACAACAAAU GUCACCAGCA CUAGCGAGAC ne

CGCUC

INFORMATION FOR SEQ ID NO:239: SEQUENCE CHARACTERISTICS: LENGTH: 41 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239: UCACUCAACC GGCACCAGAC UACGAUCAGC AUUGGCGAGU G 41 ne INFORMATION FOR SEQ ID NO:240: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240: GGGAGACAAG AAUAAACGCU CAACGAGCAA GGAACGAAUA CAAACCAGGA AACUCAGCAA CACGCGAGCA ne INFORMATION FOR SEQ ID NO:241: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241: CUCAACGACC ACAAUAACCG GAAAUCCCCG CGGUUACGGA ACACGCGAAC WO 96/40703 PCT/US96/09455 A 51 INFORMATION FOR SEQ ID NO:242: SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242: AGAAUAAACG CUCAACGAUG AGCGUGACCG AAGCUAUAAU CAGGUCGAUU CACCAAGCAA UCUUAUUCG 69 INFORMATION FOR SEQ ID NO:243: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243: ACGCUCAAAG GAUCACACAA ACAUCGGUCA AUAAAUAAGU AUUGAUAGCG ne INFORMATION FOR SEQ ID NO:244: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244: GCUCAAGCGG UCAGAAACAA UAGCUGGAUA CAUACCGCGC AUCCGCUGGG CG 52 ne INFORMATION FOR SEQ ID NO:245: SEQUENCE CHARACTERISTICS: LENGTH: 58 base pairs TYPE: nucleic acid STRANDEDNESS:. single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine PCT/US96/09455 WO 96/40703 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:245: ACCAUCUAGA GCUUCGAACC AUGGUAUACA AGGGAACACA AAAUUCGCGG AGGCUCCA 58 INFORMATION FOR SEQ ID NO:246: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:246: GGGAGACAAG AUAAACGCUC AAACAAGAGA GUCAAACCAA GUGAGAUCAG AGCGUUUAGC GCGGAAAGCA CAUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:247: SEQUENCE CHARACTERISTICS: LENGTH: 87 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:247: GGGAGACAAG AAUAAACGCU CAAAAAGACG UAUUCGAUUC GAAACGAGAA AGACUUCAAG UGAGCCCGCA GUUCGACAGG AGGCUCA 87 ne INFORMATION FOR SEQ ID NO:248: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:248: GGGAGACAAG AAUAAACGCU CAANNNNNNN NNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:249: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid PCT/US96/09455 WO 96/40703 (ii) (xi)

TAATACGACT

STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: DNA SEQUENCE DESCRIPTION: SEQ ID NO:249: CACTATAGGG AGACAAGAAT AAACGCTCAA INFORMATION FOR SEQ ID NO:250: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:250: GCCTGTTGTG AGCCTCCTGT CGAA INFORMATION FOR SEQ ID NO:251: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251: GGGAGACAAG AAUAAACGCU CAAGCCCCAA ACGCAAGCGA GCAUCCGCAA CAGGGAAGAA GACAGACGAA UGAUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:252: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252: GGGAGACAAG AAUAAACGCU CAAGCCCCAA ACGCAAGUGA GCAUCCGCAA CAGGGAAGAA GACAGACGAU UGAUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:253: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine WO 96/40703 PCT/US96/09455 188 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253: GGGAGACAAG AAAUAAACNC UCAAGCCCCA AACGCAAGUG AGCAUCCGCA ACAGGGAAGA AGACAGAUGA AUGAUUCGAC AGGAGGCUCA CAACAGGC 98 INFORMATION FOR SEQ ID NO:254: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAGACAAG

GGGAAGAAGA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:254: AAUAAACNCU CAAGCCCCAA GCAAGUGAGC AUCCGCAACA CAGACGAGUG AUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:255: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:255: GGGAGACAAG AAUAAACNCU CAAGCCCCAA ACGCAAGUGA GCAUCCGCAA CAGGGAAGAA GACAGACGAA UGAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:256: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:256: .CAAG AAUAAACGCU CAAGCAAAAG GCGUAAAUAC ACCUCCGCAA AGAA GACGCAGGGA CGGUUCGACA GGNGGCUCAC AACAGGC cytosine uracil 97

GGGAGA

CUGGGA

INFORMATION FOR SEQ ID NO:257: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid PCT/US96/09455 WO 96/40703 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:257: GGGAGACAAG AAUAAACGCU CAAACAGCUA CAAGUGGGAC AACAGGGUAC AGCGGAGAGA AACAUCCAAA CAAGUUCGAC AGGAGGCUCA CAACAGGC 98 INFORMATION FOR SEQ ID NO:258: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:258: GGGAGACAAG AAUAAACGCU CAAAUCAACU AAACAACGCA GUCACGAGAA CGACCGGKCU GACUCCGAAA GUUCGACAGG AGGCUCACAA CAGGC ne INFORMATION FOR SEQ ID NO:259: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:259: GGGAGACAAG AAUAAACGCU CAAACGAGAG CACCAAGGCA ACAGAUGCAG AAGAAGUGUG CGCGCGCGAA AUUCGACAGG AGGCUCACAA CAGGC ne INFORMATION FOR SEQ ID NO:260: SEQUENCE CHARACTERISTICS: LENGTH: 98 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:260: GGGAGACAAG AAUAAACGCU CAAUAAGACA ACGAACAGAC AGAAGCGAAA ne WO 96/40703 PCT/US96/09455 190 AAGGGGCGCC GCAGCAACAA CAAAUUCGAC AGGAGGCUCA CAACAGGC INFORMATION FOR SEQ ID NO:261: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAGACAAG

UGGAAUAGGA

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:261: AAUAAACGCU CAACGUGUAC CACAACAGUU CCACGGAAGC CGCAGAGGAA UUCGACAGGA GGCUCACAAC AGGC 94 INFORMATION FOR SEQ ID NO:262: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: (xi)

GGGAGACAAG

GGRGGRAGRC

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:262: AAUAAACGCU CAAACAAAAU UWUGGUGGGC CCCGCAACMG CGUUGAAGGC UUCGACAGGA GGCUCACAAC AGGC 94 INFORMATION FOR SEQ ID NO:263: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cyto (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 urac (xi) SEQUENCE DESCRIPTION: SEQ ID NO:263: GGGAGACAAG AAUAAACGCU CAAGAUCAUA ACGAGAGGAG AGGGAGAACU ACACGCGCGC GAGGAAAGAG UUCGACAGGA GGCUCACAAC AGGC 94 sine :il INFORMATION FOR SEQ ID NO:264: SEQUENCE CHARACTERISTICS: LENGTH: 89 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: WO 96/40703 PCT/US96/09455 191 OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH, uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:264: GGGAGACAAG AAUAAACGCU CAAACACAAA UCGGGCAGGG ACUGGGUUGG GCACGGCAGG GCGCCUUCGA CAGGAGGCUC ACAACAGGC 89 INFORMATION FOR SEQ ID NO:265: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:265: GGGAGACAAG AAUAAACGCU CAAGUGGGCU CGGGCCGGAU GUCUACGGGU GUGAAGAAAC CCCUAGGGCA GGGUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:266: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: (ix) FEATURE:RNA OTHER INFORMATION: All C's are 2'-NH 2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:266: GGGAGACAAG AAUAAACGCU CAAGAUCAGC GGAACUAAGA AAUGGAAGGC UAAGCACCGG GAUCGGGAGA AUUCGACAGG AGGCUCACAA CAGGC cytosine uracil INFORMATION FOR SEQ ID NO:267: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:267: GGGAGACAAG AAUAAACGCU CAAUAACAAA GCAGCAAAGU ACCAGAGGAG AGUUGGCAGG GUUUAGGCAG CUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:268: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs WO 96/40703 PCT/US96/09455 192 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:268: GGGAGACAGA AUAAACGCUC AAAGACCAAG GGACAGCAGC GGGGAAAAAC AGAUCACAGC UGUAAGAGGG CUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:269: SEQUENCE CHARACTERISTICS: LENGTH: 93 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:269: GGGAGACAAG AAUAAACGCU CAAAGUCGGG GAUAGAAACA CACUAAGAAG UGCAUCAGGU AGGAGAUAAU UCGACAGGNG GCUCACAACA GGC 93 ne INFORMATION FOR SEQ ID NO:270: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:270: GGGAGACAAG AAUAAACGCU CAAGAGUAUC ACACAAACCG GCACGGACUA AGCAGAAGGA GGUACGGAAG AUUCGACAGG AGGCUCACAA CAGGC INFORMATION FOR SEQ ID NO:271: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:271: GGGAGACAAG AAUAAACNCU CAACGAAAUA GAAGGAACAG AAGAAUGGBG ne ne WO 96/40703 PCTIUS96/09455 AWGNGGGAAA UGGCAACGAA UUCGACAGGN GGCUCACAAC AGGC INFORMATION FOR SEQ ID NO:272: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are (ix) FEATURE: 2'-NH 2 cytosine (xi)

GGGAGACAAG

AAAGGGAGMM

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:272: AAUAAACGCU CAAACGAGAC CCUGGAUACG AGGCUGAGGG MRRAMCUARR CKCUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:273: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:273: GGGAGACAAG AAUAAACGCU CAAGAAGGAU ACUUAGGACU ACGUGGGAUG GGAUGAAAUG GGAGAACGGG AGUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:274: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:274: GGGAGACAAG AAUAAACGCU CAAAACGCAC AAAGUAAGGG ACGGGAUGGA UCGCCCUAGG CUGGAAGGGA ACUUCGACAG GAGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:275: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: WO 96/40703 PCT/tS96/09455 OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:275: GGGAGACAAG AAUAAACGCU CAAGGUGAAC GGCAGCAAGG CCCAAAACGU AAGGCCGGAA ACNGGAGAGG GAUUCGACAG GNGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:276: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2 '-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:276: GGGAGACAAG AAUAAACGCU CAAUGAUAUA CACGUAAGCA CUGAACCAGG CUGAGAUCCA UCAGUGCCCA GGUUCGACAG GAGGCUCACA ACAGGC 96 INFORMATION FOR SEQ ID NO:277: SEQUENCE CHARACTERISTICS: LENGTH: 94 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:277: GGGAGACAAG AAUAAACGCU CAAGAUCAUA ACGAGAGGAG AGGGAGAACU ACACGCGCGC GAGGAAAGAG UUCGACAGGA GGCUCACAAC AGGC 94 ne INFORMATION FOR SEQ ID NO:278: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, cytosi] (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:278: GGGAGACAAG AAUAAACGCU CAAUCAAGUA AGGAGGAAGG GUCGUGACAG AAAAACGAGC AAAAAACGCG AGUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:279: SEQUENCE CHARACTERISTICS: LENGTH: 93 base pairs WO 96/40703 PCT/US96/09455 195 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil

GGGAGACAAG

AAAUGGCUAG

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:279: AAUAAACGCU CAAAAGGUGC CGGGUUGGAG GGGUAGCAAG GGCGCASGAU UCGACAGGNG GCUCACAACA GGC 93 INFORMATION FOR SEQ ID NO:280: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:280: CAAG AAUAAACGCU CAACCAACGC GCACCCCGCA GCAAACGAAA AGAC AGGUGCAAGA CAGUUCGACA GGAGGCUCAC AACAGGC cytosine uracil 97

GGGAGA

UUGGGG

INFORMATION FOR SEQ ID NO:281: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:281: GGGAGACAAG AAUAAACKCU CAACAAACAA UAUCGGCGCA GGAAAACGUA GAAACGAAAM GGAGCUGCGY GGAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:282: SEQUENCE CHARACTERISTICS: LENGTH: 93 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:282: GGGAGACAAG AAUAAACGCU CAAUGAUAGC ACAGUGUAUA AGAAAACGCA WO 96/40703 PCT/US96/09455 ACACCGCGCG CGGAAAGAGU UCGACAGGAG GCUCACAACA GGC INFORMATION FOR SEQ ID NO:283: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:283: GGGAGACAAG AAUAAACGCU CAAGAUCAUC GCAGUAUCGG AAUCGACCCU CAGUGGGUGA CAUGCGGACA AGUUCGACAG GAGGCUCACA ACAGGC 96 ne INFORMATION FOR SEQ ID NO:284: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:284: GGGAGACAAG AAUAAACGCU CAAGUACCGG GAAGGGAUGA ACUGGGAUAU GGGAACGGAG GUCAGAGGCA CGAUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:285: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:285: GGGAGACAAG AAUAAACGCU CAAGCAAUGG AACGCUAGGA GGGAACAUAA GCAGGGCGAG CGGAGUCGAU AGCUUCGACA GGAGGCUCAC AACAGGC 97 ne INFORMATION FOR SEQ ID NO:286: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: WO 96/40703 PCT/US96/09455 OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:286: GGGAGACAAG AAUAAACGCU CAAAACAGAA CUGAUCGGCG CAGGUUGAUA AAGGGGCAGC GCGAAGAUCA CAAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:287: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-NH 2 cytosine (xi)

GGGAGACAAG

GACGAGAAGU

OTHER INFORMATION: All U's are 2'-NH 2 uracil SEQUENCE DESCRIPTION: SEQ ID NO:287: AAUAAACGCU CAAGGGAAAC GGAAAGGGAC AAGGCGAACA AGACGGAGUA GGAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:288: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:288: GGGAGACAAG AAUAAACGCU CAANNNGAGG AAGGGCACGC AAGGAAACAA AACACAAAGC AGAAGUAGUA AGAUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:289: SEQUENCE CHARACTERISTICS: LENGTH: 95 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE:RNA (ii) (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH, (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:289: CAAG AAUAAACGCU CAAGUACRCA GUGAGCAGAA GCAGAGAGAC IUGGG AUGAAAUGGK CUUCGACAGG AGGCUCACAA CAGGC 2 cytosine Suracil

GGGAGA

UUGGGA

INFORMATION FOR SEQ ID NO:290: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs WO 96/40703 PCT/US96/09455 198 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine

FEATURE:

OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:290: GGGAGACAAG AAUAAACNCU CAACCGACGU GGACDCGCAU CGGCAUCCAG ACCAGGCUGN BCNGCACCAS ACGUUCGACA GGAGGCUCAC AACAGGC 97 INFORMATION FOR SEQ ID NO:291: SEQUENCE CHARACTERISTICS: LENGTH: 11 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-NH 2 cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-NH 2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:291: GGGAAGAAGA C 11 INFORMATION FOR SEQ ID NO:292: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F c (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F L (xi) SEQUENCE DESCRIPTION: SEQ ID NO:292: ACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNN ACUC GCCCGA :ytosine iracil 66

GGGAGG

CAGACG

INFORMATION FOR SEQ ID NO:293: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine OTHER INFORMATION: All U's are (xi) SEQUENCE DESCRIPTION: SEQ ID NO:293: 2'-F uracil WO 96/40703 PCT/US96/09455 GGGAGGACGA UGCGGGCAAA UUGCAUGCGU UUUCGAGUGC UUGCUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:294: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: (xi)

GGGAGGACGA

CUCCUCAGAC

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:294: UGCGGUGCUU AAACAACGCG UGAAUCGAGU UCAUCCACUC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:295: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: RNA (ii) (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F c (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:295: ACGA UGCGGUUAAU UCAGUCUCAA ACGGUGCGUU UAUCGAGCCA WGAC GACUCGCCCG AA :ytosine iracil 72

GGGAGG

CUGAUC

INFORMATION FOR SEQ ID NO:296: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytos: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uraci (xi) SEQUENCE DESCRIPTION: SEQ ID NO:296: ACGA UGCGGCUUAG AGCUCAAACG GUGUGACUUU CAAGCCCUCU AGAC GACUCGCCCG A 71 ine

L

GGGAGG

AUGCCC

INFORMATION FOR SEQ ID NO:297: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCTIUS96/09455 200 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:297: GGGAGGACGA UGCGGUACCU CAAAUUGCGU GUUUUCAAGC AGUAUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:298: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:298: ACGA UGCGGACCCU CAAAUAACGU GUCUUUCAAG UUGGUCAGAC CCCG A 61

GGGAGG

GACUCG

INFORMATION FOR SEQ ID NO:299: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine U's are 2'-F (xi)

GGGAGGACGA

GACUCGCCCG

SEQUENCE DESCRIPTION: SEQ ID NO:299: UGCGGACCCU CAAAUAGCGU GCAUUUCAAG CUGGUCAGAC uracil 61 A INFORMATION FOR SEQ ID NO:300: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine U's are 2'-F (xi)

GGGAAGACGA

GACUCGCCCG

SEQUENCE DESCRIPTION: SEQ ID NO:300: UGCGGCGCUC AAAUAAUGCG UUAAUCGAAU UCGCCCAGAC uracil 61 A INFORMATION FOR SEQ ID NO:301: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 201 LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:301: GGGAGGACGA UGCGGCAAAC AAGCUCAAAU GACGUGUUUU UCAAGUCCUU GUUGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:302: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:302: GGGAGGACGA UGCGGUAGUA AGUCUCAAAU GUUGCGUUUU UCGAAACACU UACAUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:303: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAGGACGA

OTHER INFORMATION: All U's are 2'-F SEQUENCE DESCRIPTION: SEQ ID NO:303: UGCGGAGACU CAAAUGGUGU GUUUUCAAGC CUCUCCCAGU uracil 62 CGACUCGCCC GA INFORMATION FOR SEQ ID NO:304: SEQUENCE CHARACTERISTICS: LENGTH: 63 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULAR TYPE: RNA (ii) (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID C's are 2'-F cytosine U's are 2'-F uracil NO:304: WO 96/40703 PCT/US96/09455 202 GGGAGGACGA UGCGGUGCUC AAAUGAUGCG UUUCUCGAAU CCACCCAGAC GACUCGCCCG AGG INFORMATION FOR SEQ ID NO:305: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:305: GGGAGGACGA UGCGGCCAUC GGUCUUGGGC AACGCGUUUU CGAGUUACCU AUGGUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:306: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:306: GGGAGGACGA UGCGGCCAUC GGUCUUGGGC AACGCGUUUU CGAGUUACCU ACAUCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:307: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:307: GGGAGGACGA UGCGGGACCC UUAGGCAACG UGUUUUCAAG UUGGUCAGAC GACUCGCCCG A 61 ne INFORMATION FOR SEQ ID NO:308: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCTIUS96/09455 203 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:308: GGGAGGACGA UGCGGACGUA GCUCUUAGGC AAUGCGUAUU UCGAAUUAGC UGUGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:309: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:309: GGGAGGACGA UGCGGAGUCU UAGGCAGCGC GUUUUCGAGC UACUCCAUCG CCAGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:310: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAAGACGA

AUCCUCAGAC

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:310: UGCGGAAUGC UCUUAGGCAG CGCGUUAAUC GAGCUAGCAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:311: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:311: GGGAGGACGA UGGGGAGUCU UAGGCAGCGC GUUUUCGAGC UACUCCAUCG CCAGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:312: SEQUENCE CHARACTERISTICS: WO 96/40703 PCTfUS96/09455 204 LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:312: ACGA UGCGGUAAUC UCUUAGGCAU CGCGUUAAUC GAGAUAGAUC AGAC GACUCGCCCG A cytosine iracil 71

GGGAGG

ACCGUC

INFORMATION FOR SEQ ID NO:313: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:313: GGGAGGACGA UGCGGCAAUG UCHCUUAGGC CACGCGUUAA UCGAGCGUGA CUGUCAGACG ACUCGCCCGA G 71 INFORMATION FOR SEQ ID NO:314: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi)

GGGAGGACGA

AUGCUCAGAC

SEQUENCE DESCRIPTION: SEQ ID NO:314: UGCGGCAUGG UCUUAGGCGA CGCGUUUAUA UCGAGUCACC GACUCGCCCG A INFORMATION FOR SEQ ID NO:315: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:315: WO 96/40703 PCT/US96/09455 GGGAGGACGA UGCGGGAUGC UUAGGCGCCG UGUUUUCAAG GCCAUCAGAC GACUCGCCCG A INFORMATION FOR SEQ ID NO:316: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:316: GGGAGGACGA UGCGGUAAUU GUCUUAGGCG CCGUGUUAUC AAGGCACAAU UUCCCUCAGA CGACUCGCCC GA 72 ne INFORMATION FOR SEQ ID NO:317: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:317: GGGAAGACGA UGCGGCUACU AGUGUCUUAG GCGGAGUGUU UAUCAAUCCA CACAUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:318: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:318: GGGAGGACGA UGCGGACUGA CUUAGGCUGC GCGCACUUCG AGCAUCAGAC GACUCGCCCG A 61 ne INFORMATION FOR SEQ ID NO:319: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCT/US96/09455 206 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi)

GGGAGGACGA

ACAUCAGACG

SEQUENCE DESCRIPTION: SEQ ID NO:319: UGCGGUGGUG UGUCUUUGGC ACCGCGUAUU UUCGAGGUAC

ACUCGCCCGA

INFORMATION FOR SEQ ID NO:320: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi)

GGGAGGACGA

ACAUCAGACG

SEQUENCE DESCRIPTION: SEQ ID NO:320: UGCGGUGGUG UGUCUUUGGC ACCGCGUAUU CUCGAGGUAC

ACUCGCCCGA

INFORMATION FOR SEQ ID NO:321: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:321: GGGAGGACGA UGCGGGCUCU UCAGCAACGU GUUAUCAAGU UAGCCCAGAC ie GACUCGCCCG A INFORMATION FOR SEQ ID NO:322: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:322: GGGAGGACGA UGCGGCGUAA CUUCAGCGGU GUGUUAAUCA AGCCUUACGC CAUCUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:323: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: (xi)

GAGGACGAUG

CUCGCCCGA

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:323: CGGGCUCUUA AGCAACGUGU UAUCAAGUUA GCCCAGACGA INFORMATION FOR SEQ ID NO:324: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:324: ACGA UGCGGUCUCA AGCAAUGCGU UUAUCGAAUU ACCGUACGCC AGAC GACUCGCCCG A :ytosine .racil 71

GGGAGG

UCCGUC

INFORMATION FOR SEQ ID NO:325: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:325: GGGAGGACGA UGCGGAAAUC UCUUAAGCAG CGUGUAAAUC AAGCUAGAUC UUCGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:326: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are (xi) SEQUENCE DESCRIPTION: SEQ ID N0:326: 2'-F uracil WO 96/40703 PCT/US96/09455 GGGAGGACGA UGCGGUUCUU AAGCAGCGCG UCAAUCGAGC UAACCCAGAC GACUCGCCCG A INFORMATION FOR SEQ ID NO:327: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:327: GGGAGGACGA UGCGGAUCUU AAGCAGCGCG UCAAUCGAGC UAACCCAGAC GACUCGCCCG AG 62 ne INFORMATION FOR SEQ ID NO:328: SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:328: ACAGCUGAUG ACCAUGAUUA CGCCAAGCUU AAGCAGCGCG UUUUCGAGCU CAUGUUGGUC AGACGACUCG CCCGA INFORMATION FOR SEQ ID NO:329: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:329: GGGAGGACGA UGCGGAGGGU CUUAAGCAGU GUGAUAAUCA AACUACUCUC CGUGUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:330: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCT/US96/09455 209 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:330: GGGAGGACGA UGCGGGAUCU UAAGCAGUGC GUUAUUCGAA CUAUCCCAGA CGACUCGCCC GA 62 INFORMATION FOR SEQ ID NO:331: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:331: GGGAGGACGA UGCGGUGCUA UUCUUAAGCG GCGUGUUUU CAAGCCAAUA UCAUCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:332: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:332: GGGAGGACGA UGCGGUCUUA AGCGGCGCGA UUUUCGAGCC ACCGCAUCCU CCGUGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:333: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:333: GGGAGGACGA UGCGGCCUCU UAAGCGUCGU GUUUUUCAAG CUGGUCAGAC GACUCGCCCG A 61 ne INFORMATION FOR SEQ ID NO:334: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F (xi) SEQUENCE DESCRIPTION: SEQ ID NO:334: 'ACGA UGCGGAUACC ACCUCUUAAG CGACGUGCAU UUCAAGUCAG AGAC GACUCGCCCG A cytosine uracil 71

GGGAGG

AUGGUC

INFORMATION FOR SEQ ID NO:335: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F i (xi) SEQUENCE DESCRIPTION: SEQ ID N0:335: ACGA UGCGGUGCUA UUCUUAAGCG GCGUGUAAAU CAAGCUAGAU rCAGA CGACUCGCCC GA cytosine iracil 72

GGGAGG

CAUCGU

INFORMATION FOR SEQ ID NO:336: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:336: GGGAGGACGA UGCGGAACGA CUCUUAAGCU GUGCGUUUUC GAACAAGUCG UAACUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:337: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are (xi) SEQUENCE DESCRIPTION: SEQ ID N0:337: 2'-F uracil WO 96/40703 PCT/US96/09455 GGGAGGACGA UGCGGCUCUC AUUUWGCGCG UAAAUCGAGC UAGCCCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:338: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:338: GGGAGGACGA UGCGGAGUCW CUCUCCACCA KCGUGUKUUA AUCAAGCUAN UGCCUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:339: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:339: GGGAGGACGA UGCGGUCUAC GGUCUCUCUG GCGGUGCGUA AAUCKAACCA GAUCGCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:340: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:340: GGGAGGACGA UGCGGUDAUU UCYUAAUCHG AGCGUUUAUC UAUCUMAAUK AUCCUCAGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:341: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA PCT/US96/09455 WO 96/40703 212 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:341: GGGAGGACGA UGCGGAUCGC AAUMUGUWGC GUUCUCKAAA CAGCCUCAGA CGACUCGCCC GA 62 INFORMATION FOR SEQ ID NO:342: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:342: GGGAGGACGA UGCGGUGGUU CUAGGCACGU GUUUUCAAGU GUAAUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:343: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:343: GGGAGGACGA UGCGGAAACA UGUGUUUUCG AAUGUGCUCU CCUCCCCAAA CAACYCCCCC AA 62 INFORMATION FOR SEQ ID NO:344: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:344: GGGAGGACGA UGCGGAAGGC CGUGUUAAUC AAGGCUGCAA UAAAUCAUCC UCCCCAGACG ACUCGCCCGA INFORMATION FOR SEQ ID NO:345: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 213 LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:345: ACGA UGCGGAGGAU CGUGUUCAUC AAGAUUGCUC GUUCUUUACU AGAC GACUCGCCCG A 71 ne

GGGAGG

GCGUUC

INFORMATION FOR SEQ ID NO:346: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:346: GGGAGGACGA UGCGGUCAAA GUGAAGAAUG GACAGCGUUU UCGAGUUGCU UCACUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:347: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:347: GGGAGGACGA UGCGGGGAGA AUGGCCAGCG UUUAUCGAGG UGCUCCGUUA ACCGGCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:348: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID C's are 2'-F cytosine U's are 2'-F uracil NO: 348: PCT[US96/09455 WO 96/40703 GGGAGGACGA UGCGGGAGGA AUGGACWGCG UAUAUCGAGU UGCCUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:349: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:349: GGGAGGACGA UGCGGAUCGA UUUCAUGCGU UUUUCGAGUG ACGAUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:350: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:350: GGGAGGACGA UGCGGAGACC CUAAGMGSGU KSUUUUCAAS CUGGUCWGAC ie GACUCGCCCG A INFORMATION FOR SEQ ID NO:351: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:351: GGGAGGACGA UGCGGUUAGC CUACACUCUA GGUUCAGUUU UCGAAUCUUC CACCGCWGAC GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:352: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCTIUS96/09455 215 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:352: GGGAGGACGA UGCGGUUAGG UCAAUGAUCU UAGUUUUCGA UUCGUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:353: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:353: GGGAGGACGA UGCGGACGUG UGUAUCRARU UUUCCGCUGU UUGUGCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:354: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:354: ACGA UGCGGACAGG GUUCUUAGGC GGAGUGUUCA UCAAUCCAAC AGAC GACUCGCCCG A cytosine uracil 71

GGGAGG.

CAUGUC

INFORMATION FOR SEQ ID N0:355: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosj (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uraci (xi) SEQUENCE DESCRIPTION: SEQ ID N0:355: ,ACGA UGCGGCGAUU UCCACAGUUU GUCUUAUUCC GCAUAUCAGA 'GCCC GA 62 ine

L

GGGAGG

CGACUC

INFORMATION FOR SEQ ID NO:356: SEQUENCE CHARACTERISTICS: WO 96/40703 PCTIUS96/09455 216 LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi: (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:356: ACGA UGCGGAUAYU CAGCUYGUGU KUUUUCDAUC UUCCCCAGAC CCCG A 61 ne

GGGAGG

GACUCG

INFORMATION FOR SEQ ID NO:357: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:357: GGGAGGACGA UGCGGCACAC GUGUUUUCAA GUGUGCUCCU GGGAUCAGAC ne GACUCGCCCG A INFORMATION FOR SEQ ID NO:358: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosir (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:358: GGGAGGACGA UGCGGCAAUG UGUUUCUCAA AUUGCUUUCU CCCUUCAGAC ie GACUCGCCCG A INFORMATION FOR SEQ ID NO:359: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID C's are 2'-F cytosine U's are 2'-F uracil NO:359: PCT/US96/09455 WO 96/40703 217 GGGAGGACGA UGCGGAUACU ACCGUGCGAA CACUAAGUCC CGUCUGUCCA CUCCUCAGAC GACUCGCCCG A INFORMATION FOR SEQ ID NO:360: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:360: ACGA UGCGGAUACU AUGUGCGUUC ACUAAGUCCC GUCGUCCCCU GGOAGG CAGACGACUC GCCCGA INFORMATION FOR SEQ ID NO:361: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: (xi)

GGGAGGACGA

UCACUCAGAC

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:361: UGCGGGUACU AUGUACGAUC ACUAAGCCCC AUCACCCUUC NACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:362: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:362: GGGAGGACGA UGCGGUUACU AUGUACAUUU ACUAAGACCC AACGUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:363: SEQUENCE CHARACTERISTICS: LENGTH: 72 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA PCTIUS96/09455 WO 96/40703 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:363: GGGAGGACGA UGCGGUUWCU AUGUWCGCCU UACUAAGUAC CCGUCGACUG UCCCAUCAGA CGACUCGCCC GA 72 INFORMATION FOR SEQ ID NO:364: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:364: GGGAAGACGA UGCGGUGUUG AUCAAUGAAU GUCCUCCUCC UACCCCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:365: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:365: GGGAGGACGA UGCGGUGUUU GUCAAUGUCA UGAUUAGUUU UCCCACAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:366: SEQUENCE CHARACTERISTICS: LENGTH: 64 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:366: GGGAGGACGA UGCGGCGGUC UUAAGCAGUG UGUCAAUCAA ACUAUCGUCA GACGACUCGC CCGA 64 INFORMATION FOR SEQ ID NO:367: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 219 LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:367: GGGAGGACGA UGCGGUUCUU AAGCAGCGCG UCAAUCGAGC UAACCCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:368: SEQUENCE CHARACTERISTICS: LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:368: GGGAGGACGA UGCGGAAUGR CCCGUUACCA WCAAUGCGCC UCDUUGMCCC CAAACAACYC CCCCAA 66 INFORMATION FOR SEQ ID NO:369: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:369: GGGAGGACGA UGCGGAAUYU CGUGYUACGC GUYYYCUAUC CAAUCUACCC CMUCUCCAAU CAGACGACYC INFORMATION FOR SEQ ID NO:370: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:370: WO 96/40703 PCT/US96/09455 220 GGGAGGACGA UGCGGCGCUU ACAAUAAUUC UCCCUGAGUA CAGCUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:371: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:371: GGGAGGACGA UGCGGAACUU CUUAGGCAGC GUGCUAGUCA AGCUAAGUUC CACCUCAGAC GACUCGCCCG A 71 ne INFORMATION FOR SEQ ID NO:372: SEQUENCE CHARACTERISTICS: LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi] (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:372: GGGAGGACGA UGCGGCACAA UCUUCGGCAG CGUGCAAGAU CAAGCUAUUG UUGUCAGACG ACUCGCCCGA ne INFORMATION FOR SEQ ID NO:373: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:373: GGGAGGACGA UGCGGUCAUU AACCAAGAUA UGCGAAUCAC CUCCUCAGAC 51 GACUCGCCCG A 6: ne INFORMATION FOR SEQ ID NO:374: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCT/US96/09455 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:374: GGGAGGACGA UGCGGUCAUU CUCUAAAAAA GUAUUCCGUA CCUCCACAGA CGACUCGCCC GA 62 INFORMATION FOR SEQ ID NO:375: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:375: GGGAGGACGA UGCGGGUGAU CUUUUAUGCU CCUCUUGUUU CCUGUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:376: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAGGACNA

CCCCUCAGAC

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:376: UGCGGUCUAG GCAUCGCUAU UCUUUACUGA UAUAAUUACU GACUCGCCCG A 71 INFORMATION FOR SEQ ID NO:377: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: (xi)

GGGAGGACGA

GACUCGCCCG

OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:377: UGCGGAGUWW GCNCGGUCCA GUCACAUCCW AUCCCCAGAC A 61 INFORMATION FOR SEQ ID NO:378: SEQUENCE CHARACTERISTICS: WO 96/40703 PCT/US96/09455 LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosil (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:378: :ACGA UGCGGCUCUC AUAUKGWGUR UUYUUCMUUC SRGGCUCAAA CCCC AA 62 ne

GGGAGG

CAAYYC

INFORMATION FOR SEQ ID NO:379: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi2 (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:379: GGGAGGACGA UGCGGCUUGU UAGUUAAACU CGAGUCUCCA CCCCUCAGAC ne GACUCGCCCG A INFORMATION FOR SEQ ID NO:380: SEQUENCE CHARACTERISTICS: LENGTH: 62 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:380: GGGAGGACGA UGCGGUCUCU WCUVACVUGU RUUCACAUUU UCGCYUCAAA CAACYCCCCC AA 62 ne INFORMATION FOR SEQ ID NO:381: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:381: WO 96/40703 PCT/US96/09455 223 GGGAGGACGA UGCGGUURAC AAUGRSSCUC RCCUUCCCWG GUCCUCAGAC GACUCGCCCG A 61 INFORMATION FOR SEQ ID NO:382: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:382: AGGAGGACGA UGCGGUUAUC UGAARCWUGC GUAAMCUARU GUSAAASUGC AACRACRAAC AACYCSCCCA A 71 ne INFORMATION FOR SEQ ID NO:383: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosi (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:383: AGGAAGACGA UGCGGUUCGA UUUAUUUGUG UCAUUGUUCU UCCAUCAGAC GACUCGCCCG A 61 ne INFORMATION FOR SEQ ID NO:384: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: OTHER INFORMATION: All (xi) SEQUENCE DESCRIPTION: SEQ ID GUGAUGACAU GGAUUACGCC AGACGACUCG CCCGA INFORMATION FOR SEQ ID NO:385: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: C's are 2'-F cytosine U's are 2'-F uracil NO:384: WO 96/40703 PCT/US96/09455 224 OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:385: UGCGUGUUUU CAAGCA 16 INFORMATION FOR SEQ ID NO:386: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:386: CUCAAAUUGC GUGUUUUCAA GCA 23 INFORMATION FOR SEQ ID NO:387: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID N0:387: GGUACCUCAA AUUGCGUGUU UUCAAGCAGU AUC 33 INFORMATION FOR SEQ ID NO:388: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE: (xi)

GGAGUCUUAG

OTHER INFORMATION: All SEQUENCE DESCRIPTION: SEQ ID GCAGCGCGUU UUCGAGCUAC UCC U's are 2'-F uracil NO:388: 33 INFORMATION FOR SEQ ID NO:389: SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA WO 96/40703 PCTIUS96/09455 225 (ix) FEATURE: OTHER INFORMATION: All C's are 2'-F cytosine, (ix) FEATURE: (xi)

GGGAGGACGA

NN NNNCAGAC OTHER INFORMATION: All U's are 2'-F uracil SEQUENCE DESCRIPTION: SEQ ID NO:389: UGCGGNNNNN NNNNNNNNNN NNNNNN NNNNN'NNNNN 51 GACUCGCCCG A 7: INFORMATION FOR SEQ ID NO:390: SEQUENCE CHARACTERISTICS: LENGTH: 97 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA (ix) FEATURE: OTHER INFORMATION: All (ix) FEATURE: C's are 2'-F cytosine (xi)

GGGAGACAAG

NNN NN OTHER INFORMATION: All U's are 2'-F1 SEQUENCE DESCRIPTION: SEQ ID NO:390: AAUAAACGCU CAANNNNNNN NNNNNNN NNThNNNNNNN NNNINNNNN NNNUUCGACA GGAGGCUCAC AACAGGC i1racil

Claims (54)

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

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

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

4. The method of Claim 3 wherein said single-stranded nucleic acids are ribonucleic acids. The method of Claim 4 wherein said nucleic acids comprise modified ribonucleic acids.

6. The method of Claim 5 wherein said nucleic acids comprise modified ribonucleic acids selected from the group consisting of 2'-amino NH2) modified ribonucleic acids and 2'-fluoro modified ribonucleic acids.

7. The method of Claim 3 wherein said single-stranded nucleic acids are deoxyribonucleic acids.

8. The method of Claim 2 further comprising e) forming a multivalent Complex comprising two nucleic acid ligands identified in step c). WO 96/40703 PCTfUS96/09455 227

9. The method of Claim 5 further comprising e) substituting 2'-O-methyl ribonucleic acids for 2'-OH ribonucleic acids in the nucleic acid ligands identified in step c). The method of Claim 1 wherein said lectin is selected from the group consisting of a mammalian lectin, a plant lectin, a microbial lectin and a viral lectin.

11. The method of Claim 1 wherein said lectin is wheat germ agglutinin.

12. The method of Claim 1 wherein said lectin is a selectin.

13. The method of Claim 12 wherein said selectin is selected from the group consisting of L-selectin, E-selectin, and P-selectin.

14. The method of Claim 1 wherein said lectin is serum mannose binding protein. A purified and isolated non-naturally occurring nucleic acid ligand to a lectin.

16. The nucleic acid ligand of Claim 15 which is a non-naturally occurring nucleic acid ligand having a specific binding affinity for said lectin, such lectin being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by said lectin.

17. The nucleic acid ligand of Claim 15 wherein said lectin is selected from the group consisting of a mammalian lectin, a plant lectin, a microbial lectin and a viral lectin.

18. The nucleic acid ligand of Claim 15 wherein said lectin is selected from the group consisting of wheat germ agglutinin, L-selectin, E-selectin and P- selectin. WO 96/40703 PCT/US96/09455 228

19. The nucleic acid ligand of Claim 15 wherein said lectin is wheat germ agglutinin. The nucleic acid ligand to wheat germ agglutinin of Claim 19 wherein said nucleic acid ligand is a ribonucleic acid (RNA) ligand.

21. The nucleic acid ligand of Claim 20 which comprises a modified ribonucleic acid.

22. The nucleic acid ligand of Claim 21 wherein said modified ribonucleic acid is a 2'-amino (NH 2 modified ribonucleic acid.

23. The nucleic acid ligand to wheat germ agglutinin of Claim 22 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2.

24. The nucleic acid ligand of Claim 23 wherein said ligand is selected from the group consisting of SEQ ID NOS: 4-55. The nucleic acid ligand of Claim 20 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 56-63.

26. The nucleic acid ligand to wheat germ agglutinin of Claim 19 wherein said ligand is substantially homologous to and has substantially the same ability to bind said wheat germ agglutinin as a ligand selected from the group consisting of the sequences set forth in Table 2.

27. The nucleic acid ligand to wheat germ agglutinin of Claim 19 wherein said ligand has substantially the same structure and the same ability to bind said wheat germ agglutinin as a ligand selected from the group consisting of the sequences set forth in Table 2.

28. The nucleic acid ligand of Claim 15 wherein said lectin is a selectin.

29. The nucleic acid ligand of Claim 28 wherein said selectin is selected from the group consisting of L-selectin, E-selectin and P-selectin. WO 96/40703 PCT/US96/09455 229

30. The nucleic acid ligand of Claim 29 wherein said selectin is L- selectin.

31. The nucleic acid ligand to L-selectin of Claim 30 wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand.

32. The nucleic acid ligand of Claim 31 which comprises a modified ribonucleic acid.

33. The nucleic acid ligand of Claim 32 wherein said modified ribonucleic acid is selected from the group consisting of a 2'-amino (2'-NH 2 modified ribonucleic acid and a 2'-fluoro modified ribonucleic acid.

34. The nucleic acid ligand to L-selectin of Claim 33 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 8 and 16. The nucleic acid ligand of Claim 34 wherein said ligand is selected from the group consisting of SEQ ID NOS: 67-117 and 293-388.

36. The nucleic acid ligand of Claim 31 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 118-125.

37. The nucleic acid ligand to L-selectin of Claim 30 wherein said ligand is substantially homologous to and has substantially the same ability to bind said L- selectin as a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.

38. The nucleic acid ligand to L-selectin of Claim 30 wherein said ligand has substantially the same structure and the same ability to bind said L-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 8, 12 and 16.

39. The nucleic acid ligand to L-selectin of Claim 30 wherein said nucleic acid ligand is deoxyribonucleic acid (DNA). WO 96/40703 PCT[US96/09455 230

40. The nucleic acid ligand to L-selectin of Claim 39 wherein said ligand is an DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 12.

41. The nucleic acid ligand of Claim 40 wherein said ligand is selected from the group consisting of SEQ ID NOS: 129-180 and 185-196.

42. The nucleic acid ligand of Claim 39 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NOS: 181-184.

43. The nucleic acid ligand of Claim 29 wherein said selectin is P- selectin.

44. The nucleic acid ligand to P-selectin of Claim 43 wherein said nucleic acid ligand is ribonucleic acid (RNA) ligand. The nucleic acid ligand of Claim 44 which comprises a modified ribonucleic acid.

46. The nucleic acid ligand of Claim 45 wherein said modified ribonucleic acid is selected from the group consisting of a 2'-amino (2'-NH 2 modified ribonucleic acid, a 2'-fluoro modified ribonucleic acid, and a Methyl modified ribonucleic acid.

47. The nucleic acid ligand to P-selectin of Claim 46 wherein said ligand is an RNA ligand selected from the group consisting of the nucleotide sequences set forth in Tables 19, 21 and

48. The nucleic acid ligand of Claim 47 wherein said ligand is selected from the group consisting of SEQ ID NOS: 199-219 and 236-290.

49. The nucleic acid ligand of Claim 44 wherein said ligand comprises sequences selected from the group consisting of SEQ ID NO: 291. The nucleic acid ligand to P-selectin of Claim 43 wherein said ligand is substantially homologous to and has substantially the same ability to bind said P- 231 selectin as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and

51. The nucleic acid ligand to P-selectin of Claim 43 wherein said ligand has substantially the same structure and the same ability to bind said P-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and

52. The nucleic acid ligand to P-selectin of Claim 46 wherein said nucleic acid ligand is deoxyribonucleic acid (DNA).

53. The nucleic acid ligand of Claim 15 wherein said ligand is a ribonucleic acid ligand.

54. The nucleic acid ligand of Claim 53 which comprises a modified 20 ribonucleic acid. The nucleic acid ligand of Claim 54 wherein said modified ribonucleic acid is selected from the group consisting of 2'-amino (2'-NH 2 modified ribohucleic acids, 2'-fluoro modified ribonucleic acids and 25 Methyl modified ribonucleic acids.

56. The nucleic acid ligand of Claim 15 wherein said ligand is a deoxyribonucleic acid. 0 30 57. The nucleic acid ligand of Claim 15 wherein said ligand has been chemically modified at the sugar and/or phosphate and/or base.

58. A multivalent Complex comprising a plurality of ligands of Claim

59. A nucleic acid ligand to a lectin identified according to the method comprising: a) contacting a candidate mixture of nucleic acids with a lectin, wherein nucleic acids having an increased affinity to said lectin relative to the S candidate mixture may be partitioned from the remainder of the candidate mixture; Sb) partitioning the increased affinity nucleic acids from the 0 remainder of the candidate mixture; and WO 96/40703 PCTIUS96/09455 232 c) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said lectin, whereby nucleic acid ligands of said lectin may be identified.

60. A method for treating a lectin-mediated disease comprising administering a pharmaceutically effective amount of a nucleic acid ligand to a lectin.

61. The method of Claim 60 wherein said nucleic acid ligand to a lectin is identified according to the method of Claim 1.

62. The method of Claim 60 wherein said lectin is a selectin.

63. The method of Claim 62 wherein said selectin is L-selectin.

64. The method of Claim 62 wherein said selectin is P-selectin.