CA2223275A1 - High affinity nucleic acid ligands to lectins - Google Patents

Abstract

This invention discloses high-affinity oligonucleotide ligands to lectins, specifically nucleic acid ligands having the ability to bind to the lectins, wheat germ agglutinin, L-selectin, E-selectin and P-selectin. Also disclosed are the methods for obtaining such ligands.

Description

wo 9~0703 PCT/US9C~0915' High Af~lnity Nucleic Acid Ligands to Lectins FIELD OF THE ~VENlION
Described herein are methods for identifying and ~lepalirIg high-affinity 10 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 T .~ n~Ts by EXponential enrichment.
Specifically tlic~losed herein are high-affinity nucleic acid ligands to wheat germ ~vvllltinin (WGA), L-sçlçctin~ E-seIectin, and P-selectin.
BACKGROUND OF I~lk INVENTION
The biological role of lectins (non e.~yl~alic carbohydrate-binding proteins of non-immllne origin; I. J. Goldstein et al., 1980, Nature 285:66) is inextricably linked to that of carbohydrates. One function of carbohy~d~es is the mo~lifi/~tion of 20 physical characteristics of glyco-conjugates (i.e., 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-con~ugates are information rich molecl-les (N.
Sharon and H. Lis, 1989, Science 246:227-~34; K. Drickamer and M. Taylor, 25 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 (i.e., the~re are lectins that bind omy galactose or ~i-acetyl~.al~tose; other lectins bind mannose; still others bind sialic acid and so on; K Dtick~mer and M. Taylor, supra). Specificity of binding enables lectins to decode information contained in the carbohydrate 30 portion of glyco-conjugates and thereby mP~Ti~te many i~ ol~nt biological functions.
~ lC~O~S m~mm~Ti~n, plant, rnicrobial and viral lechns have been described (I. Of ek and ~. Sharon, 1990, Current Topics in Microbiol.ar,d Immunol. 151:91-113; K. Drickamer and M. Taylor, supra; I. J. Goldstein and R. D. Poretz, 1986, in 35 The T ecrinc, p.p. 33-247; A. Varki, supra). These proteins m~ te a diverse array of biological processes which include: trafficking of lysosomal enzy~nes, clearance of serum proteins, endocytosis, phagocytosis, opsonization, microbial and viral infections, toxin binding, fertilization, irnmllne and infl~mm~tory responses, cell adhesion and migration in development and in pathological conditions such as 40 metastasis. Roles in symbiosis and host defense have been proposed for plant W O ~6/4~703 PCT~US96/09455 5 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 div~ y and importance of processes m~ ted by lectins is illustrated by two well tiocllm~nt~A m~rnm~ lectins, the asialoglycoprotein receptor and theserum mannose binding protein, and by the viral lectin, influenza virus 10 hema~,gluLi~ . The hepatic asialoglycoprotein receptor specifically binds g~ tQse and N-~ety~ rtose and thereby m~Ai~tt c the clearance of serum glyco~oteins thatpresent t~rmin~l N-acetylg~ tose or gal~tose rçcidllec, exposed by the prior removal of a t~rmin~l sialic acid. The human m~nnose-binding protein (~P) is a serum protein that binds termin~l mannose, fucose and N-acetylgll~cQs~
15 rec~ s These t~rmin~l residues are cornmon on microbes but not m~mm~ n glyco-conjugates. The binding specificity of MBP conctit~ltçs a non-i~l.l"~
m~çh~nicm for distinguishing self from non-self and m~ t~s host defense through opso!,i7i1lion and complement fixation. Tnflll~n7~ virus hemagglutinin m~ t~s the initial step of infection, ~ .,t to nasal epith~ l cells, by binding sialic ~id 20 residues of cell-surface receptors.
The divcl~iLy of lectin m~rli~t~d functions provides a vast array of potential lllcld~culic targets for lectin antagonists. Both lectins that bind endogenous carbohyJldtes and those that bind exogenous carbohydrates are target c~n~ tt-s For çY~mrle, antagonists to the m~rnm~ n selectins, a family of endogenous 25 carbohydrate binding lectins, may have thcl~pcuLic applir~tionc in a variety of leukocyte-m~ tecl disease states. Inhibition of selectin binding to its receptorblocks cellular adhesion and conse~uently may be useful in treating infl~mm~tion, coagulation, transplant rejection, tumor metastasis, rh~ m~toid arthritis, reperfusion injury, stroke, myocardial infarction, burns, psoriasis, multiple sclerosis, bacterial 30 sepsis, hypovolaemic and trallm~tic shock, acute lung injury, and ARDS.
The selectins, E-, P- and L-, are three homologous C-type lectins that recognize the tetrasacch~ri(le7 sialyl-LewisX (C. Foxall et al, 1992, J. Cell Biol.
117,895-902). Selectins m~ te the initial adhesion of neullu~hils and monocytes to activated vascular endothelium at sites of infl~mm~tion (R. S. Cotran et al., 1986, 35 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 m~osPnt~ric lymph nodes (W. M.
Gallatin et al., 1983, Nature 304,30; T. K. Kichimoto et al., 1990, Proc. Natl.
40 Acad. Sci. 87,2244-) and P-selectin m.o~ tes the adherence of platelets to neutrophils and monocytes (S-C. Hsu-Lin et al., 1984, J. BioL Chem. 259,9121).

W O g~'2~703 PCTrUS96/09455 Selectin antagonists (antibodies and carbohydrates) have been shown to block the extravasation of neutrophils at sites of infl~mm~tjon (P. Piscueta and F.
W. Luscinskas, 1994, Am. J. Pathol. 145, 461-469), to be efficacious in animal models of ischemia/reperfusion (A.S. 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 (M.S. Mulligan et al., 1993, J. Immunol. 151, 6410-6417; A. Seçk~mp et al., 1994, Am. J. Pathol. 144, 592-598), insulitis/diabetes (X.D. Yang et al., 1993, Proc. Natl. Acad. Sci. 90,10494-10498), mpningitis (C. Granet et al., 1994, J.
Clin. Invest. 93, 929-936), hemorrhagic shock (R.K. Winn et al., 1994, Am J.
Physiol. Heart Circ. Physiol. 267, H2391-H2397) and transplantation. In addition, selectin expression has been docnm~nte~l in models of arthritis (F. Jamar et al., 1995, Radiology 194, 843-850), exp~ tz~l allergic encephalomyelitis (J.M.
Dopp et al., 1994, J. Neuloi~ lunol. 54, 129-144), cutaneous infl~mm~tion (A.
Siber et al., 1994, Lab. Invest. 70, 163-170) glomeruloneph,iLis (P.G. Tipping et al., 1994, Kidney Int. 46, 79-88), on leukaemic cells and colon carcinomas (R.M.Lafrenie et al., 1994, Eur. J. Cancer [A] 30A, 2151-2158) and L-selectin receptors have been observed in myelin~tr~l regions of the central nel ~ous system (K. Huang et al., 1991, J. Clin. Invest. 88, 1778-1783). These animal model data strongly support the ~pe~ ion of a thtl~ell~ic role for selectin antagonists in a wide variety of disease states in which host tissue damage is neutrophil-m~ te~
Other examples of lectins that recogI~ize endogenous carbohydrates are CD22,13, CD23, CD44 and sperm lectins (A. Varki, 1993, Glycobiol.3, 97-130;
P.M. W~cs~rm~n, 1988, Ann. Rev. Biochem. 57, 415-442). CD22~ is involved in early stages of B lymphocyte activation; antagonists may mo~ te the illlrll.l.l~response. CD23 is the low affinity IgE receptor; antagonists may modulate the IgE
30 response in allergies and asthma. CD44 bindc hyaluronic acid and thereby mrtli~t~s cell/cell and cell/matrix adhesion; antagonists may modulate the infl~mm~tory response. Sperm lectins are thought to be involved in sperm/egg a&esion and in the acrosomal response; antagonists may be effective contraceptives, either by blocking adhesion or by intlnring a premature, spermicidal acrosomal response.
Antagonists to lectins that recognize exogenous carbohydrates may have wide application for the prevention of infectious ~lise~ces- Many viruses (influenza A, B and C; Sendhi, Newcastle disease, coronavirus, rotavirus, encephalomyelitisvirus, enchephalomyocarditis virus, reovirus, paramyxovirus) use lectins on the surface of the viral particle for ~tt~rhmP~nt to cells, a prerequisite for infection;
40 antagonists to these lectins ale expected to prevent infection (A.Varki, 1993, Glycobiol.3, 97-130). Similarly colonizationlinfection strategies of many bacteria WO 9'~ 7~3 PCT/U~g~ C5 S utilize cell surface lectins to adhere to m~mm~ n cell surface glyco-conjugates.
Antagonists to b~rtPri~l cell surface lectins are expected to have theld~euLic potential for a wide spe~ m of b~rtpri~l infections, including: gastric (Helicobacter pylori), urinary tract (E. coli), pulmonary (Klebsiella pneumoniae, Stretococcus pne~moni~P-~ Mycoplasma pnp~lmoniae) and oral (Actinomyces n~Pslu~-li and 10 Actinomyces viscosus) colonization/infection (S.N. Abraham, 1994, Bacterial hPsinc, in The Handbook of Immunoph~rm~ology: Adhesion Molecules, C.D.
~egner, ed; B.J. Mann et al., 1991, Proc. Natl. Acad. Sci. 88, 3248-3252). A
specific b~tPri~l mP~i~tPA disease state is Pseudomonas aeruginosa infection, the leading cause of morbidity and mortality in cystic fibrosis patiPnts The expectation 15 that high affinity antagonists will have efficacy in treating P. aeruginosa infection is based on three observations. First, a bacterial cell surface, GalNAc~1~Gal binding lectin mPAi~tPs infection by adherence to asialogangliosides (aGM1 and aGM2) of pulmonary epithPlinm (L. Tmlmdo et al., 1995, Proc. Natl. Acad. Sci 92,3019-3023). Second, in vitro, t'ne binding of P. aeruginosa is competed by the 20 gangliosides' tetr~c~cçh~ri~P moiety, Gal~1-3GalNAc~l~Gal~l~Glc. Third, in vivo, in~till~tion of antibodies to Pseudomonas surface ~ntigçnc can prevent lung and pleural damage (J.F. Pittet et al., 1993, J. Clin. Invest. 92, 1221-1228).
Non-b~rtPri~l microbes that utilize lectins to initiate infection include ~nt~moeba histalytica (a Gal specific lectin that mP~j~tPs adhesion to intestinal 25 mucosa; W.A. Petri, Jr., 1991, AMS News 57:299-306) and Plasmodium facip~.u~l (a lectin specific for the terminal NeuSAc(a2-3)Ga~ of glycophorin A of erthrocytes; P.A. Orlandi et al., 1992, J. Cell Biol. 116:901-909). Antagonists to these lectins are potential th~ldl~c;ulics for dysentery and m~
Toxins are another class of proteins that recognize exogenous carbohydrates 30 (K-A Karlsson, 1989, Ann. Rev. Biochem. 58:309-350). Toxins are complex, two domain molecules, composed of a functional and a cell recognitionladhesion domain. The adhesion domain is often a lectin (i.e., bacterial toxins: pertussistoxin, cholera toxin, heat labile toxin, verotoxin and tetanus toxin; plant toxins: ricin and abrin). Lectin antagonists are expected to prevent these toxins from binding35 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 incnffi~ient levels of this lectin suffer from severe infections, but adults do not. The high frequency of mutations in 40 both oriental and Canc~ci~n populations suggests a condition may exist in which low levels of serum mannose-binding protein are advantageous. Rheumatoid arthritis 5 (RA) may be such a condition. The severity of RA is correlated with an increase in IgG antibodies lacking tennin~l g~l~rtQse 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 irnmunocomplexes may contribute 10 to host tissue darnage through complement activation. Similarly, the eosinophil basic protein is cytotoxic. If the cytotoxicity is mr~ t~o(l by the lectin activity of this protein, then a lectin antagonist may have therapeutic applications in treating eosu,ophil mr~ t~cl lung damage.
Lectin antagonists may also be useful as im~gin~ agents or diagnostics. For 15 ry~mrle~ E-selectin antagonists may be used to image infl~mrd endothrlillm Similarly antagonists to specific serum lectins, i.e. m~nnose-binding protein, may also be useful in ~lu~l~tit~ g protein levels.
Lectins are often complex, multi-(lom~in, mllltimrric proteins. However, the carbohydrate-binding activity of m~mm~ n lectins is norm~lly the ~ro~clLy of a 20 carbohy~ e recognition domain or CRD. The CRDs of ."~....,.~li~n lectins fall into three phylogençtir~lly conselved classes: C-type, S-type and P-type (K. Drirl~m~r and M.E. Taylor, 1993, Annu. Rev. Cell Biol. 9, 237-264). C-type lectins requireCa~ for ligand binding, are extr~c~ r membrane and soluble proteins and, as a class, bind a,variety of carbohydrates. S-type lectins are most active under re~llring 25 conditions, occur both intra- and extracellularly, bind ,B-galactosides and do not require Ca~. P-type lectins bind mannose 6-phosphate as their primary ligand.
Although lectin specificity is usually expressed in terrns of monos~rch~rides and/or oligosacçhr~ s (i.e., MBP binds mannose, fucose and N-acetylglllcos~",;".o), the affinit,v for monos~cch~rides is weak. The dissociation 30 constants for monomeric s~rch~rides are typically in the millimolar range (Y.C. 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 (W.I. Weis et 35 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 monos~rch~nde binding; N-acetylglucosarmine 40 has equatorial 3- and 4-hydroxyls while fucose has similarly configured hydroxyls at the 2 and 3 positions.

CA 0222327=, l997-l2-02 W O g~ 3 PCT/U~ 91~5 The affinity of the mannose-binding protein and other lectins for their natural ligands is greater than that for monos~c~h~ricles Increased specificity and affinity can be accomplich~d by est~bliching additional contacts b~lweell a protein and its ligand (K. Dric~m~r, 1993, supra) either by 1) additional contacts with the termin~l sugar (i.e., chicken hepatic lectin binds N-acetylglucose amine with greater afflnity than mannose or fucose suggesting interaction with the 2-s~lbstinlt-nt); 2) clustering of CRDs for binding complex oligos~r~h~ritles (i.e., the m~mm~ n asialylglycoplotei,l receptor); 3) interactions with additional saccharide residues (i.e., the lectin domain of selectinc appears to interact with two residues of the tetrasaccharide sialyl-LewisX: with the charged termin~l residue, sialic acid, and 15 with the fucose residue; wheat germ ag~luLi~ appears to interact with all three residues of trimers of N-acetylglucos~mine); or by 4) contacts with a non-carbohydrate portion of a glyco-protein.
The low affinity of lectins for mono- and oligo-s~çh~rides presents major ~liffiClllti~S in developing high affinity antagonists that may be useful ther~peuti~s.
20 Approaches that have been used to develop antagonists are similar to those that occur in nature: 1) addition or modification of substihlentc to increase the number ofinte~ ions; and 2) mlll L;- - Irl ;,~l ion of simple lig~n-ls The first approach has had limited success. For example, homologues of sialic acid have been analyzed for aff1nity to influenza virus h~m~g~ tinin (S.J.
25 Watowich et al. 1994, Structure 2:719-731). The dissociation constants of the best analogues are 30 to 300 yM which is only 10 to 100-fold better than the standardmonosaccharide .
Modifications of carbohydrate ligands to the selectins have also had limited success. In static ELISA competition assays, sialyl-Lewisa and sialyl-LewisX have 30 ICsos of 220 ~LM and 750 ~lM, respectively, for the inhibition of the binding of an E-selectin/lgG chimera to immobilized sialyI-LewisX (R.M. Nelson et al., 1993, J.
Clin. Invest. 91, 1157-1166). The ICso 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 !lM. Similarly, removal of the N-acetyl from sialyl-35 lewisX improves inhibition in an assay dependent manner (C. Foxall et al., 1992, J.Cell Biol. 117, 89~-902; S.A. DeFrees et al., 1993, J. Am. Chem. Soc. 115, 7549-7550).
The second approach, mllltim~ri7~tion of simple lig~n-ls, can lead to dramatic improvements in affinity for lectins that bind complex carbohydrates (Y.C. Lee, 40 supra). On the other hand, the approach does not show great enhancement for lectins that bind simple oligosaccharides; dimerizing sialyl-LewisX, a minim~l CA 0222327~ 1997-12-02 WO 9~0703 PCT/US96/09455 5 carbohydrate ligand for E-selectin, improves inhibition approximately 5-fold (S.A.
DeFrees et al., supra).
An ~It~m~tive approach is to design compounds that are chernic~lly unrelated to the natural ligand. In the static ELISA cornretition assays inositol polyanions inhibit L- and P-selectin binding with ICsos that range from 1.4 !lM to 2.8 mM (O.
10 Cecconi et al., 1994, J. Biol. Chem. 269, 15060-15066). Synthetic oligopeptides, based on selectin amino acid sequences, inhibit neull~,phil binding to immobilized P-selectin with IC50S ranging from 50 ~LM 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 SFT F~X
15 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 ~ffinities that are several orders of m~gnitll~e greater than that of the co~ g sugar can be obtained by the a~lo~liate manipulation of the nucleic acid ligand to colllpetiLol ratio. Since the carbohydrate 20 binding site is the active site of a lectin, essentially all ligands i~ol~t~d by this procedure will be antagonists. In addition, these SELEX ligands will exhibit much greater spe~ifi~ity than mono~P~ ;c and oligomeric sacrh~ es.
A method for the in vitro evolution of nucleic acid molecules with highly specific bind~ing to target molecules has been developed. This method, Systematic 25 Evolution of T.ig~n~l~ by EXponential enrichment, termed SELEX, is described in United States Patent Application Serial No. 07/536,428, entitled "syslem~tir Evolution of T igantl.c by Exponential Fnrichm~nt~ now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled "NucleicAcid r.ig~nflc," now United States Patent Number 5,475,096, United States Patent30 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 filn~l~mPnt~lly novel method for making a nucleic acid ligand to any35 desired target molecule.
The SELEX method involves selection from a mixture of c~n~ tP
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 rnixture of nucleic acids, preferably 40 comprising a segment of randomized se~uence, the SELEX method includes steps of contacting the ~ ulG with the target under conditions favorable for binding, WO 96t40703 PCTAU53C~'~g1',5 5 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-.~nri~hç~ Lule of nucleic acids, then leitel~Ling the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield 10 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 SFT T X in conjunction with gel electrophoresis to 15 select nucleic acid mol~c~ s with specific structural charact~rictics~ such as bent DNA. United States Patent Application Serial No. 08/123,935, filed Sept~ bel 17,1993, entitled "Photoselection of Nucleic Acid T .ig~ntlc" describes a SELEX based method for selecting nucleic acid ligands co"lil;";"g photoreactive groups capable of binding and/or photocrosclinkin~ to and/or photoinactivating a target molecule.
20 United States Patent Application Serial No. 08/134,028, filed October 7, 1993, entitled "High-Affinity Nucleic Acid T ig~n-lc That Dic-~rimin~tç Between Theophylline and Caffeine," describes a method for identifying highly specific nucleic acid ligands able to riiccrimin~te between closely related molecules, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed 25 October 25, 1993, entitled "Systematic Evolution of Ligands by Expone~ti~l Fnrichm~nt 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 Proclll~ing Nucleic Acid Ligands" describes 30 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 EXponenti~l Enricnment:
Chemi-SELEX," describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the i~lentific~tion of high-affinity nucleic 35 acid ligands cont~ining 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-i~entified nucleic acid ligands col-t~i ni l~g modified nucleotides are described in United States Patent Application Serial No.
40 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid Ligands Cont~ining Modified Nucleotides," that describes oligonucleotides cont~inin~

W O 96/40703 PCTAJS9G~'~S'S' nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines.
United States Patent Application Serial No. 08/134,0281 supra, describes highly specific nucleic acid ligands cont~ining one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States Patent Application Serial No. 08/264,029, filed June 22, 1994, entitled "Novel Method of ~aldtion of 2' Modified Pyrimifline Intr~molec~ r Nucleophilic Dicpl~t~em~ nt " describes novel methods for making 2'-modified nucleosides.
The SELEX method encomp~cses combining selecte-l oligonucleotides with other s~.lecte~l oligonucleotides as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled "Systematic Evolution of T igantl~ by Exponential F.nril l""~ Chim~ric SELEX". The SELEX method also inrllldec 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 "System~tic Evolution of T .ig~n~ls by Expon~nti~l Fnn~h~n~ 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 ~lo~lLies, and the effirient ~mplifi~tion 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-immllne origin, which include, but are not lirnited to, those described above.
Specifically, high affinity nucleic acid ligands to wheat germ aggllltinin, and various selectin proteins have been isolated. For the purposes of t'ne invention the terrns wheat gerrn agglutinin, wheat gerrn lectin and WGA are used interchangeably.
Wheat gerrn agglulinill (WGA) is widely used for isolation, purification and structural studies of glyco-conjugates. As outlined above, the selectins are irnportant anti-infl~mm~tQry targets. Antagonists to the selectins modulate extravasion of leukocytes at sites of infl~mm~tion and thereby reduce neutrophil caused host tissue darnage. Using the SELEX technology, nigh affinity antagonists of L-selectin, E-selectin and P-selectin mP~i~t~d adhesion are isolated.

WO9~'4~703 PCT/U',CI~S~

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 identifi~ and produced. More particularly, nucleic acid ligands are provided that are capable of binding specifically to Wheat Germ ~gy,llllinil~ (WGA), L-Selectin, E-selectin and P-selectin.
Further inf~ d~l in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to lectins comrnsing the steps of (a) plepaling a c~nflid~te mixture of nucleic acids, (b) partitioning belweell members of said c~n~ te ll~u~ e on the basis of affinity to said lectin, and (c) amplifying the selected molecules to yield a mixture of nucleic acids enri~h~d for nucleic acid1~ se~uences with a relatively higher affinity for binding to said lectin.
More ~pecifi~lly, the present invention in~ es the nucleic acid ligands to lectins i-lentified according to the above-~les~riked method, in~ lin~ those ligands to Wheat Germ AggluLi~ l 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 25.Additionally, nucleic acid ligands to E-selectin and serum m~nnose binding protein are provided. Also in~ d are nucleic acid ligands to lectins that are subst~nti~lly homologous to any of the given ligands and that have sll~st~nti~lly 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 ~orm as the ligands presented herein and that have subst~nti~lly 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 the~dl~eu~ic, prophylactic and diagnostic applications.

BREF DESCRIPTION OF THE FIGURES
Figure l shows consensus hairpin secondary structures for WGA 2 -NH2 - 3~ RNA ligands: (a) fa~uly 1, (b) family 2 and (c) family 3. Nucleo~ide 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/U' X/~3 155 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 ex~c~ ent in which the binding of 5 nM 32P-labeled F14.12 (SEQ ID NO: 78) to PBMCs (107/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 COlllp~LiLOl).
Figure S shows the results of a co.ll~eLi~ion e~ ullent in which the binding of 5 nM 32P-labeled F14.12 (SEQ ID NO: 78) to PBMCs (107/ml) is co..lyeted 15 with increasing concentr~tions of the blocking monoclonal anti-L-selectin antibody, DREG-56, or an isot~ye m~t~h~A, negative control antibody. RNA Bound equals 100 x (net counts bound in the presence of co" IlJcLiLo. /net counts bound in the absence of comretitor).
Figure 6 shows the results of a Col-lyeLiLi~le ELISA assay in which the 20 binding of soluble LS-Rg to immobilized sialyl-LewisXtBSA conjugates is competed with increasing conce~ dtions 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 (OD4so in the presence of co~ )e~ilor)/(OD450 in the ~hsence of colll~e~ilor). The observed OD4so was corrected for nonspecific binding by subtracting the OD4so in the absence of LS-Rg from the e~r. ;~ t~l values. In the absence of comretitor the OD4so 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 Ca~/~Ig~ with 4 mM EDTA reduced the OD4so to 0.045.
Figure 7 shows hairpin secondary structures for representative L-selectin 2'NH2 RNA ligands: (a) F13.32 (SEQ. ~D NO: 67), family I; (b) 6.16 (SEQ. ID
NO: 84), family m; and (c) 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 schem~tic representation of each dimeric and " lu~ ;c oligonucleotide complex: (a) dimeric branched oligonucleotide, (b) multivalent streptavidin/bio-oligonucleotide complex (A: s~eptavidin; B: biotin); (c) dimeric dumbell olir,onucleotide; (d) dimeric fork oligonucleotide.
Figure 9 shows binding curves for the L-selectin SELEX fifteenth round ssDNA pool to PBMCs (107/ml).
Figure 10 shows the results of a competition experiment in which the binding of 2 nM 32P-labeled round 1~ ssDNA to PBMCs (107/ml) is competed with wo s~c ~03 PCT/US96/09455 5 increasing conce~ ations of the blocking monoclonal anti-L-selectin antibody, DREG-56, or an isotype m~t- h~cl, negative control antibody. RNA Bound equals 100 x (net counts bound in the presence of competitor/net counts bound in the absence of co~ ctitor).
Figure 11 shows L-selectin specific binding of LD201T1 (SEQ ID NO: 185) 10 to human lymphocytes and granulocytes in whole blood. a, FITC-LD201T1 bindingto lymphocytes is colllpctcd by DREG-56, unlabeled LD201T1, and inhibited by EDTA. b, FITC-LD201T1 binding to granulocytes is competed by DREG-56, nnl~led LD201T1, and inhibited by EDTA. All s~mplc s were stained with 0.15 mM FITC-LD201T1; thick line: FITC-LD201T1 only; thick dashed line: FITC-15 LD201T1 with 0.3 mM DREG-56; mc-t'inm thick line: ~llC-LD201T1 with 7 mM
lml~ l NX280; thin line: FITC-LD201T1 stained cells, reassayed after addition of 4 mM EDTA; thin dashed line: autofluorcsccllce.
Figure 12 shows the concenCllc hairpin secondary ~ u.;lulcs for farnily 1 ssDNA ligands to L-selectin. Nucleotide seqllfAnc-e is in standard one letter code.
20 Invariant nucleotides are in bold type. The base pairs at highly variable positions are ~çsi n~tc-1 N-N'. To the right of the stem is a matrix showing the number of occul~ces of particular base pairs for the position in the stem that is on the same line.
Figure 13 shows that in vitro pre-tre~tm~nt of human PBMC with NX288 25 (SEQ ID NO: 193) inhibits lymphocyte trafficking to SCID mouse PLN. Human PBMC were purified from hep~rinice-l blood by a Ficoll-Hypaque gradient, washed twice with HBSS (c~lci~lm/m~gnc--sium free) and labeled with 5 lCr (Amersham).
After labeling, the cells were washed twice with HBSS (col-t~ g c~lc-illm and m~gnC~sium) and 1% bovine serum albumin (Sigma). Female SCID mice (6-12 30 weeks of age) were injected hllldvellously with 2X106 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 euth~nice~ PLN, MLN, Peyer's patches, spleen, liver, lungs, thymus, kidneys and bone marrow were 35 removed and the counts incorporated into the organs ~etçrrnined by a Packard gamma counter. Values shown represent the mean + s.e. of triplicate samples, andare representative of 3 ~e~ ellts.
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 40 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 CA 02223275 l997-l2-02 WO 9~"C7~3 PCT/U~ 5155 5 with 2xl06 cells. One to 5 min prior to injecting the cells, the animals were injected with either 15 pmol DREG-56 or 4 nmol mo~lifie-l oligonucleotide. Animals were sc~rifice~l 1 hour after injection of cells. Counts incorporated into organs were qll~ntified as descri'oed in Figure 13. Values shown represent the mean + s.e. of triplic~tç s~mrles~ and are representative of 2 e~e~ e~
Figure 15 shows the concenslls 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 ~le~cign~t~tl N-N'. To the right of the stem is a matrix showing the number of oc~;ul~lces of particular base pairs for the position in the stem that is on the same 15 line.
Figure 16 shows the concen~uc 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 decign~t~d N-N'. To the right of the stem is a matrix showing the number of 20 oc~ ces of particular base pairs for the position in the stem that is on the same line.

DETAILED DESCRIPTION OF THE ~VENTION
This,application describes high-affinity nucleic acid ligands to lectins 25 identified through the method known as SELEX. SELEX is described in U.S.
Patent Application Serial No. 07/536,428, entitled "Systematic Evolution of T .ig~ntls by EXponential Enrichment", now abandoned; U.S. Patent Application Serial No.
07/714,131, filed June 10, 1991, entitled "Nucleic Acid T ig~n(ls", now United States Patent No. 5,475,096; United States Patent Application Serial No.
30 07/931,473, filed August 17, 1992, entitled "Nucleic Acid T.ig~ncls", now United States Patent No. 5,270,163, (see also PCT/US91/04078). These applications, each specifically inc~Jlpol~lL~d herein by reference, are collectively called the SELEX
Patent Applications.
In its most basic form, the SELEX process may be defined by the following 35 series of steps:
1) A candidate mixture of nucleic acids of differing sequence is prepared.
The c~n~id~te mixture generally inc~ es regions of fixed sequences (i.e., each of the mtombers of the c~n-lid~t~ mixture contains the same sequences in the same location) and regions of randornized sequences. The fixed sequence regions are 40 selected either: (a) to assist in the ~mr)lifi~tion steps described below, (b) to mimic S a sequence known to bind to the target, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the c~ntli~l~tt~. mixture. The randor~ized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randornized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 10 percent).
2) The c~ntlid~tP rnixture is contacted with the sçlectecl target under conditions favorable for binding bclwecll the target and members of the c~n-liA~mixture. Under these ci~ n~ n~es, the interaction between the target and the nucleic acids of the c~n~ te mixture can be considered as forming nucleic acid-15 target pairs between the target and those nucleic acids having the strongest affinityfor 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) 20 c~llcs~ollding to the highest affinity nucleic acids exist in the c~n~lid~tç rnixture, it is generally desirable to set the partitioning criteria so that a .signifiç~nt amount of the nucleic acids in the c~nAi~l~te mixture (approximately .05-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively 25 higher aff~ity to the target are then amplified to create a new c~ntliA~t~ mixture that iS .onrich~l in nucleic acids having a relatively higher affinity for the target.
S) By repeating the partitioning and arnplifying steps above, the newly formed c~n~ te lllib~lulc contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase.
30 Taken to its extreme, the SELEX process will yield a candidate mixture cont~inin,, one or a small number of unique nucleic acids represçnting those nucleic acids from the originai c~n~ te mixture having the highest affinity to the target molecule.The SELEX Patent Applications describe and elaborate on this process in great detail. Tn~ rl are targets that can be used in the process; methods for 3~ partitioning nucleic acids within a c~n~liA~te. mixture; and methods for amplifyinc partitioned nucleic acids to generate çnrich~cl c~n~liA~te mixture. The SELEX Patent Applications also describe ligands obtained to a number of target species, in~lu~lina 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 40 chemical modifications are made in order to increase the in vivo stability of the ligand or to enhance or m~ tç the delivery of the ligand. Examples of such W O 96/40703 PCT/U~ 3'-' 5 modifications include chemical substitutions at the sugar and/ or phosphate and/or base positions of a given nucleic acid sequence. See, e.g., U.S. Patent Application Serial No. 08/117,991, filed September 9, 1993, entitled "High Affinity Nucleic Acid T .ig~n~ls Cont~ining Modified Nucleotides" which is specifically incorporated herein by lefel~nce. Additionally, in co-pending and commonly ~ssign~l U.S.
10 Patent Application Serial No. 07/964,624, filed October 21, 1992 ('624), now U.S.
Patent No. 5,496,938, methods are described for obtaining illlp~oved nucleic ~idligands after SELEX has been performed. The '624 application, entitled "Methods of Producing Nucleic Acid T ig~nllc," is specifically inco,~ola~ed herein by reference. ~urther included in the '624 patent are methods for del~,. .,.il~ing the three-15 tlim~rl~ional structures of nucleic acid li~anll$ Such methods include m~t~m~tir,~lmodeling and structure morlifi~tions of the SELEX-derived li~an-lc, such as ch~mir~l morlifir~tion and nucleotide substit~-tio~ Other motlifir,~tions are known to one of o,dinaly skill in the art. Such morlifir~tions may be made post-SELEX
(modification of previously j~l~ntifiçcl unmodified ligands) or by incol~ol~ion into 20 the SELEX process. Additionally, the nucleic acid ligands of the invention can be complexed with various other compoullds, inrl~l-lin,, but not lirnited to, lipophilic colllpo~ulds or non-imm--nogenic, high molecular weight compounds. Lipophilic compounds inrllldç, but are not lirnited to, cholesterol, dialkyl glycerol, and diacyl glycerol. Non-immunogenic, high molecular weight colll~oullds include, but are 25 not limited to, polyethylene glycol, dextran, albumin and m~gn~tit~. The nucleic acid ligands described herein can be complexed with a lipophilic compound (e.g., cholesterol) or ~tt~rh~ri to or e~c~rsnl~te-l in a complex compri~ed of lipOphiliC
components (e.g., 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 ~id 30 ligands to an intr~re~ r target. The complexed nucleic acid ligands can also have çnh~nred pharmacokint-tics and stability. Urlited 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 plepa,i,lg a therapeutic or diagnostic complex comprised of a nucleic acid ligand and a lipopnilic 35 compound or a non-immunogenic, high molecular weight compound.
The methods described herein and the nucleic acid ligands identifi~l by such methods are useful for both therapeutic and diagnostic purposes. Therapeutic uses include the tre~tm~ont or prevention of diseases or m~(lic~l conditions in humanpatients. Many of the therapeutic uses are described in the background of the 40 invention, particularly, nucleic acid ligands to selectins are useful as a~llti-inft~rnm~ory agents. Antagonists to the selectins modulate extravasion of WO 96/40703 Pcr/uS96/09455 5 leukocytes at sites of infl~mm~rion and thereby reduce neutrophil caused host tissue damage. Diagnostic utilization may include both in vivo or in vitro rli~nostic applications. The SELEX method generally, and the specific adaptations of the SELEX method taught and cl~im~-l herein specifically, are particularly suited for diagnostic appli~tionc SELEX identifies nucleic acid ligands that are able to bind 10 targets with high affinity and with surprising specificity. These characteristics are, of course, the desired ~,~ellies 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 accoldi"g to any number of techniques employed by those 15 skilled in the art. Diagnostic agents need only be able to allow the user to identify the ~csence 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 posihve 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 inco~o~dte a labeling tag 20 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 selectin~
described herein may specifically be used for idel,tiri~lion of the lectin proteins.
SELEX provides high affinity ligands of a target molecule. This represents a singular achievement that is unprece~-nted in the field of nucleic acids research. The 2~ present invention applies the SELEX procedure to lectin targets. Sperific~lly~ the present invention describes the i~entifi~tion of nucleic acid ligands to Wheat Germ .A",llltinin, and the selectins, specifically, L-selectin, P-selectin and E-selectin. In the Example section below, the e~eli"le"tal 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 ph~ el~tical, it is preferred that the nucleic acid ligand ( 1 ) binds to the target in a manner capable of achieving the desired effect on the target; (2) be as small as possible to obtain the desired effect; (3) be as stable as possible; and (4) be a specific ligand to the chosen target. In most situations, it is preferred that the nucleic acid ligand have the highest 3~ possible affinity to the target.
In the present invention, a SELEX experiment was pelrolllled in search of nucleic acid ligands with specific high affinity for Wheat Germ Agc,luLil~ill from a degenerate library cont~ining 50 random positions (SON). This invention includesthe specific nucleic acid ligands to Wheat Gerrn Agglu~ l shown in Table 2 (SEQ
ID NOS: 4-55), identified by the methods described in Exarnples 1 and 2.
Specifically, RNA ligands cont~ining 2'-NH2 modified pyrimidines are provided.

CA 0222327~ 1997-12-02 WO g"~C7-,3 PCT/US96/09455 5 The scope of the ligands covered by this invention extends to all nucleic acid ligands of Wheat Germ Agglutinin, modified and unrnodified, idPntifie~l according to theSELEX procedure. More specifically, this invention incllld.os nucleic acid sequences that are s~kstantially homologous to the ligands shown in Table 2. By substanti~lly homologous it is meant a degree of primary sequence homology in excess of 70%, 10 most preferably in excess of 80%. A review of the sequence homologies of the ligands of Wheat Germ Ag~,luLinill shown in Table 2 shows that sequences with little or no primary homology may have substantially the sarne ability to bind Wheat Germ ~Ag,~ ") For these reasons, this invention also in~ dPs nucleic acid ligands that have subst~nti~lly the same ability to bind Wheat Germ ~"1, .l; "; ,- as the 15 nucleic acid ligands shown in Table 2. S~lb~ "lially the same ability to bind Wheat Germ A~14~;ll;n 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 detPrrninP whether a given sequence ~ lly homologous to those specifically described herein--has s~lbstanti~lly the same 20 abilitytobindWheatGerm ~g~ ,l;";,.
In the present invention, SELEX e~ IFl~t~ were ~ rolllled in search of nucleic acid ligands with specific high affinity for L-selectin from de~ c~
libraries cont~ining 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 25 (SEQ ID NOS: 67-117, 129-180, 185-196 and 293-388), identified by the methodsdescribed in Examples 7, 8, 13, 14, 22 and 23. Specif1cally, RNA ligands cons~ining 2'-NH2 or 2'-F pyrimi~linPs 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.
30 More specifically, this invention in~hl~Ps nucleic acid sequences that are substantially homologous to the ligands shown in Tables 8, 12 and 16. By subst~ntially 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 35 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 acidligands 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 L-selectin means that the affinity is within a few orders of ma~nih-~e of the affinity of 40 the ligands described herein. It is well within the skill of those of ordinary skill in WO 96/40703 PCT/U'~ 2 5 the art to detenninç whether a given sequence -- sl~bst~nti~lly homologous to those speçifir~lly described herein -- has substantially the sarne ability to bind L-selectin.
In the present invention, SELEX e~L~filllents were pelroll.led in search of nucleic acid ligands with specific high affinity for P-selectin from degenerate libraries cont~ining 50 random positions (SON). This invention includes the specific 10 nucleic acid ligands to P-selectin snown in Tables 19 and 25 (SEQ ID NOS: 199-247 and 251-290), id~ntifie-l by the methods described in Examples 27, 28, 35 and 36. Specifically, RNA ligands cont~ining 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-se-lectin, modified and unmodified, j~lPntifiç-l according to the 15 SELEX procedure. More specifically, this invention inrllltles nucleic acid sequences that are substantially homologous to the ligands shown in Tables 19 and 25. By subst~nti~lly 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 20 sequences with little or no primary homology may have suhst~nti~lly the sarne ability to bind P-selectin For these reasons, this invention also inrhldes nucleic acid ligands that have s~lbst~nti~lly 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 m~gnitll~e of the affinity of the 25 ligands described herein. It is well within the skill of those of ordinary skill in the art to deterrnine 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 pc;lrulllled in search of nucleic acid ligands with specific high affinity for E-selectin from a degenerate 30 library cont~ining 40 random positions (40N). This invention in~lucl~s specific nucleic acid ligands to E-selectin identified by the methods described in Example 40.
The scope of the ligands covered by this invention extends to all nucleic acid ligands of E-selectin, modified and unmodified, i~lentified according to the SELEX
procedure.
AdditionalIy, the present invention inrlndes multivalent Complexes compricing the nucleic acid ligands of the invention. The mulivalent Complexes increase the binding energy to facilit~te better binding ~ffinitjes through slower off-rates of the nucleic acid ligands. The multivalent Complexes may be useful at lower doses than their monomeric coulltel~alL~. In addition, high molecular weight 40 polyethylene glycol was included in some of the Complexes to decrease the in vivo WO ~ C7~3 PCT/U' ,c~'~n, 1;5 5 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 ph~rm~rellti~lc This invention, t_elcfolc, also inrlll-les a method for treating lectin-m~Ai~ted fiice~ces by 10 ~lminictration of a nucleic acid ligand capable of binding to a lectin.
Th~ld~culic compositions of the nucleic acid ligands may be a lminic~red parenterally by injection, although other effective ~1minictration forms, such as intraarticular injection, inh~l~nt mists, orally active form~ tions, tr~ncdt-rm~iontophoresis or suppositories, are also envisioned. One preferred carrier is 15 physiological saline solution, but it is co~temrl~tt~d that other ph~rm~relltir~lly acceptable carriers may also be used. In one prcfe,lcd embotlim~nt it is envisioned that the carrier and the ligand constitute a physiologically-compatible, slow release fqrmlll~tion. The plilll~r solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other ph~rm~cologically-20 acceptable eYri~ientC for modifying or " ,~ g the pH, osmolarity, viscosity,clarity, color, sterility, stabilit,v, rate of dissolution, or odor of the formlll~tion Similarly, the carrier may contain still other ph~rm~ologically-~rcept~hl~ ex~ipientc for modifying or m~int~inin, the stability, rate of dissolution, release, or absorption of the ligand. Such excipients are those substances usually and customarily 25 employed to formlll~te dosages for parental a~minictration in either unit dose or multi-dose form.
Once the therapeutic composition has been form~ t~l it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophili7ed powder. Such form~ tions may be stored either in a ready to use form30 or requiring reconstitution imm~ tely prior to ~Aminictration. The manner of ~tlministt~ing formul~tions cont~ining nucleic acid ligands for systemic delivery may be via subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectalsuppository.
Well established animal models exist for many of the disease states which are 35 candidates for selectin antagonist therapy. Models available for testing the efficacy of oligonucleotide selectin antagonists include:
1) mouse models for peritoneal infl~mm~tjon (P. Pizcueta and F.W.
Luscinskas, 1994, Am. J. Pathol. 145, 461-469), diabetes (A.C. H~nnin~n et al., 1992, J. Clin. Invest. 92, 2509-2515), lymphocyte trafficking (L.M. Bradley et al., 40 1994, J. Exp. Med., 2401-2406), glomerulonephritis (P.G. Tipping et al., 1994, Kidney Int. 46, 79-88), experimental allergic encephalomyelitis ( J.M. Dopp et al., CA 0222327~ 1997-12-02 WO9~ 703 PCT/US~ ~S'-5 5 1994, J. Neuroimmunol. 54: 129-144), acute infl~mm~ion in human/SCID mouse chimera (H.-C. Yan et al., 1994, J. Immunol. 152, 3053-3063), endotoxin-m.o~ t~ infl~mm~tion (W.E. Sanders et al., 1992, Blood 80, 795-800);
2) rat models for acute lung injury (M.S. Milligan et al., 1994, J. Imrnunol.
152, 832-840), hind limb ischemia/reperfusion injury (A. Seekamp et al., 1994, 10 Am. J. Pathol 144, 592-598), remote lung injury (A. Seekamp et al., 1994, supra;
D.L. Carden et al., 1993, J. Appl. Physiol 75, 2529-2543), neutrophil rolling onmesenteric venules (K. Ley et al., 1993, Blood 82, 1632-1638), myocardial infarction icch~rni~ reperfusion injury (D. Altavilla et al., 1994, Eur. J. Pharmacol.
Environ. Toxicol. Pharmacol. 270, 45-51);
3) rabbit models for hemorrhagic shock (R.K. Winn et al., 1994, Am. J.
Physiol. Heart Circ. Physiol. 267, H2391-H2397), ear ischemia reperfusion injury(D. Mihelcic et al., 1994, Bollod 84, 2333-2328) neutrophil rolling on mesentt ~c venules (A.M. Olofsson et al., Blood 84, 2749-2758), exp~rim~nt~l m~nin, itis (C.
Granert et al., 1994, J. Clin. Invest. 93, 929-936); lung, peritoneal and 20 subcutaneous bacterial infection (S.R. Sharer et al., 1993, J. Immunol. 151, 4982-4988), myocardial ischemiafrepefusion (G. Montrucchio et al., 1989, Am. J.
Physiol. 256, H1236-H1246), central nervous system i~ch~mic injury (W.M. Clark et al., 1991, Stroke 22, 877-883);
4) cat models for myocardial infraction ischemia repçrfilsion injury 25 (M.Buerke et al., 1994, J. Pharmacol. Exp. Ther. 271, 134-142);
5) dog models for myocardial infarction ischemia reperfusion injury(D.J.
Lefer et al., 1994, Circulation 90, 2390-2401);

- 6) pig models for arthritis (F. Jamar et al., 1995, Radiology 194, 843-850);

7) rhesus monkey models for cutaneous infl~mm~tion (A. Silber et al., Lab.
30 Invest. 70, 163-175);

8) cynomolgus monkey models for renal transplants (S.-L. Wee, 1991, Transplant. Prod. 23, 279-280); and 9) baboon models for dacron grafts (T. Palabrica et al, 1992, Nature 359, 848-851), septic, traumatic and hypovolemic shock (H. Redl et al., 1991, Am. J.
35 Pathol. 139, 461466).
The nucleic acid ligands to lectins described herein are useful as ph~nn~e~ltir~lc and as diagnostic reagents.
Examples The following examples are illustrative of certain emboflimtonts of the 40 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 g~ 3 PCT/U~

Wheat Gerrn AggluLinill. Exarnples 7-12 described j(l~ntific~tion and characterization of 2'-NH2 RNA ligands to L-selectin. Examples 13-21 describe identifir~tion and chal~cte, ;7~tion of ssDNA ligands to L-selectin. Exarnples 22-25 describe identification and char~ ;7~ion of 2'-F RNA ligands to L-selectin.
Example 26 describes iclentifir~tion of ssDNA ligands to P-selectin Exarnples 27-39 describes i~lentifir~tion and characterization of 2'-NH2 and 2'-F RNA ligands to P-selectin. Example 40 describes i~l~ntifir~tion of nucleic acid ligands to E-selectin.
Exarnple 1 Nucleic Acid T i~antlc to Wheat Germ Ag~,luLinill The c~ nt~l procedures outlined in this Example were used to identify and ch~d~ ize nucleic acid ligands to wheat germ ag~ (WGA) as desrribe~
in F.lr~mrles 2-6.
F.~ ..tal Procedures A) Materials Wheat Germ Lectin (Triticum vulgare) Sepharose 6MB beads were purchased from ph~ ri~ Biotech. Wheat Germ Lectin, Wheat Germ ~gCl~
and WGA are used i,lLc.~hallgeably herein. Free Wheat Germ Lectin (Triticum vulgare) and all other lectins were obtained from E Y Labuldtolies; methyl-a-D-mannopyranoside was from Calbiochem and N-acetyl-D-glucosamine, GlcNAc, and the tric~rrh~ride N N' N'-triacetylchitotriose, (GlcNAc)3, were purchased from Sigma Ch~mir,~l Co. The 2'-NH2 modified CTP and UTP were prepared according to Pieken et. al. (1991, Science 253:314-317). DNA oligonucleotides were synth~si7~ by Operon. All other reagents and chemicals were purchased from commercial sources. Unless otherwise indicated, e~ tc utilized Hanks' Balanced Salt Solutions (HBSS; 1.3 mM Cacl27 5.0 rnM KCl, 0.3 mM KH2po4~
0.5 mM MgC12.6H2O, 0.4 mM MgSO4.7H2O, 138 rnM NaCl, 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 gerrn agglutinin SELEX e~cfilllellt, the DNA template for the initial RNA pool contained 50 random nucleotides, flanked by N9 5' and 3' fixed regions (50N9) 5' gggaaaagcg~ c~ cac~ga-50N-gcuccgccagag~cc~cgagaa 3' (SEQ ID NO: 1). All C and U have 2~-NH2 substituted for 2'-OH for ribose. The primers for the PCR were the following: 5'Prirner 5' taatacgactc~ct~t~gggaaaagcg~t~t~rac~ga 3' (SEQ ID NO: 2) and 3' Primer 5' ttctcggttggtctctggcggagc 3' (SEQ ID NO: 3). The fixed regions of the WO 96/40703 PCT/U~9 ~ ''S

5 starting random pool include DNA primer ~nn~lin~ sites for PCR and cDNA
synthesis as well as the concçn~llc T7 promoter region to allow in vitro transcription. These single-stranded DNA molecules were converted into double-stranded transcribable templates by PCR ~mplific~tion. PCR conditions were 50 mMKCl, 10 mM Tris-Cl, pH 8.3, 0.1% Triton X-100, 7.5 mM MgC12, 1 mM of each 10 dATP, dCTP, dGTP, and dTTP, and 25 U/ml of Taq DNA polymerase.
Transcription reactions contained S mM DNA template, S units/~Ll T7 RNA
polymerase, 40 mM Tris-Cl (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM
sperrnicline~ 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-32P 2'-OH ATP.
The strategy for partitioning WGA/RNA complexes from unbound RNA
was 1) to in~llb~t.o the RNA pool with WGA imrnobilized 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 incuh~ting the washed beads in buffer cont~inin~ high concentrations of (GlcNAc)3. The SELEX protocol is 20 outlined in Table 1.
The WGA density on Wheat Germ Lectin Sepharose 6MB beads is approximately 5 mg/ml of gel or 116 ~M (m~nllf~rtllrer's specifications). After extensive washing in HBSS, the immobilized WGA was incubated with RNA at room te~ el~LLu~ for 1 to 2 hours in a 2 rnl siliconized column with constant rolling 25 (Table 1). 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 l; rounds 14) 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 30 HBSS (Table 1). The pel~;elllage 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 rninutes in 1% SDS, 2% ~-mercaptoethanol and extracted with phenoVchloroform. RNA
35 was reverse transcribed into cDNA by AMV reverse transcriptase at 48 ~ C for 60 rnin in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6 mM Mg(OAC)2, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/,ul 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.

W O 96/40703 PCT/U'3~ 5'~

5 D) Nitrocçlllllose Filter Binding Assay As tlescri-hed in SET FX Patent Applications, a nitroc~ ulose filter partitioning method was used to d~ ç the affinity of RNA ligands for WGA and for other proteins. Filter discs (nitrocelluloselcellulose acetate mixed matrix, 0.45 ~m pore size, Millipore; or pure nitrocellulose, 0.45 ~lm pore size, Bio-Rad) were 10 placed on a vacuum manifold and washed with 4 ml of HBSS buffer under vacuum.rtion ~ cs, colll;~il-;..g 32p labeled RNA pools and unl~h~o~ed WGA, were inrllh~t~cl in HBSS for 10 min at room te~ a~ filtered, and then imm~ tçly washed with 4 ml HBSS. The filters were air-dried and counted in a Beckm~n LS6500 liquid sr-intill~tion counter without fluor.
WGA is a homodimer, molecular weight 43.2 kD, with 4 GlcNAc binding sites per dimer. For affinity c~lr~ tionc, we assume one RNA ligand binding siteper monomer (two per dimer). The monomer concentration is defined as 2 times thedimer collce~ àtion. The equilibrillm dissociation coll~,~lt, Kd, for an RNA pool or specific ligand that binds monoph~cir~lly is given by the equation Kd = [Pf][Rfll[Rp]
where, [RfJ = free RNA concentration [Pf~ = free WGA monomer c~n~ .~ion [RP]= concel~ ~ion of RNA/WGA monomer comrl~Y~s Kd = dissociation constant A rea~np~ml-nt of this equation, in which the fraction of RNA bound at equilibrium is expressed as a function of the total concentration of the re~ct~ntc, was used to c~ tç Kds of monophasic binding curves:
q = (PT + RT + Kd - ((PT + RT + Kd) - 4 PT RT) q = fraction of RNA bound [PT] = total WGA monomer col~ellL~aLion [RT~ = total RNA concentration Kds were ~l~tPrmin~rl by least square fithng of the data points using the graphics program K~lçi~l~graph (Synergy Software, Reading, PA).
E) Cloning and Seqllçn- ing The sixth and eleventh round PCR products were re-amplified with primers which contain a BamH1 or a EcoR1 restriction endonncle~ce recognition site. Using these restriction sites the DNA sequences were inserted directionally into the pUC 18 40 vector. These recolllbillant pl~cmitls were transformed into E. coli strain JM109 (Stratagene, La Jolla, CA). Plasmid DNA was prepared according to the ~lk~linP

WO ~"0703 Pcr~3G~ r~

5 hydrolysis method (Zhou et al., 1990 Biotechniqlles 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) Colllpclilive Binding Studies Com~eLilive binding eA~f -; " ,~nts were performed to ~et~-rmine if RNA
ligands and (GlcNAc)3 bind the same site on WGA. A set of reaction nllAlules ct7nt~inin,o, a 32p labeled RNA ligand and unlabeled WGA, each at a fixed concentration (Table 5), was incubated in HBSS for 15 _in at room telll~e~dlulc with (GlcNAc)3. Individual reaction ll~u~Lulcs were then in.~llb~t~ with a 15 (GlcNAc)3 dilution from a 2-fold flillltion series for 15 minlltes The final (GlcNAc)3 concentrations ranged from 7.8 ~LM to 8.0 rnM (Table 5). The reaction mixtures were filtered, processed and counted as described in "Nitrocellulose Filter Binding Assay," paragraph D above.
Co~tiLion titration c~ ntc were analyzed by the following equation to 20 determine the conceL,~rdLion of free protein [P] as a function of the total concentration of competitQr added, [CT]:

O = [P]( l+KL[LT]/( l+KL[p])+Kc[cT]l( l+KC[P]))-PT

25 where LT is the concentration of initial ligand, KL is the binding constant of species L to the protein (~ssllming 1:1 stiochiometry) and KC is the binding colls~ t ofspecies C to the protein (~csllming 1:1 stiochiometry). Since it is difficult to obtain a direct solution for equation 1, iteration to determine values of [P] to a precision of lx10-l5 was used. Using these values of [P], the concentration of protein-ligand30 complex [PL] as a function of [CT] was det~rmin~l by the following equation:

[PL] = KL[LT][P](l+KL[P]) Since the eA~e,;~ nt~l data is expressed in terms of %[PL], the c~leul~te~
35 concentration of [PL] was norrnalized 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 (M) and minimllm (B) %[PL] was allowed to float during the analysis as shown in the following equation.
%[PL] = [PL]/[PLo]*(M-B)+B

WO g6/40703 PCT/US~ 3~1~S

S 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 colllpetition 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 f1Xed.

G) Inhibition of WGA .A~gllltin~ting, Activity ~lutin~tion 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 m~ te(l ~glntin~tion can be inhibited by sugars with appro~liate specifici~.
Visual assay of the hem~ ltin~tin~ activity of WGA and the inhibitory activity of RNA li~ntl.~, GlcNAc and (GlcNAc)3 was made in Falcon round bottom 96 well microtiter plates; using sheep erythrocytes. Each well contained 54 ~Ll of erythrocytes (2.5 x 108 cells/ml) and 54 ~11 of test solution.
To titrate WGA ~ tin~ting activity, each test solution contained a WGA
dilution from a 4-fold dilution series. The final WGA concentrations ranged from0.1 pM to 0.5 ~M. For inhibition assays, the test solutions contained 80 nM WGA
(monomer) and a dilution from a 4-fold dilution series of the desi n~t~rl inhibitor.
Reaction mixtures were inrnb~t~1 at room t~ eldtule for 2 hours, after which time no changes were observed in the precipitation ~dL~ s of erythrocytes. These t~ were carried out in Gelatin Veronal Buffer (0.15 mM CaC12, 141 mM
NaCl, 0.5 mM MgC12, 0.1% gelatin, 1.8 mM sodium barbital, and 3.1 mM
barbituric acid, pH 7.3 -7.4; Sigma #G-6514).
In the ~bsen~e of ~ggllltin~tion, erythrocytes settle as a compact pellet.
Aggl~-tin~t~d cells form a more diffuse pellet. Consequently, in visual tests, the diarneter of the pellet is diagnostic for aggllltin~tion. The inhibition e~pf,; 1 ~ Irl 1l~
inrluclecl positive and negative controls for ag~,luLination and a~lo~liate controls to show that the inhibitors alone did not alter the normal pl~ci~itation pattern.
Exarnple 2 RNA Ligands to WGA
A. SELEX
The starting RNA library for SELEX, randomized 50N9 (SEQ ID NO: 1), contained approximately 2 x 1ols molecules (2 nmol RNA). The SELEX protocol is outlined in Table 1. Binding of randomized RNA to WGA is lmtl~tect~ble at 36 ,uM WGA monomer. The dissociation constant of this interaction is estimated to be >4rnM.

CA 0222327~ 1997-12-02 wo 96/40703 Pcrlu~ 5'1~S'~5 The percentage of input RNA eluted by (GlcNAc)3 increased from 0.05 % in the first round, to 28.5 % in round 5 (Table 1). The bulk Kd of round S RNA was 600 nM (Table 1). Since an additional increase in spe~ifi¢~lly eluted RNA was not observed in round 6a (Table 1), round 6 was repeated (Table 1, round 6b) with two modifications to increase the stringency of selection: the volume of gel, and hence 10 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 percell~age of specifi~lly eluted RNA increased from 5.7 % in round 6b to 21.4 % in round 8, with continue~ l()velllent 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 buIk 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.
20 To monitor the progess of SELEX, ligands were cloned and sequenced from round6b and round 11. From each of the two rounds, 36 randomly picked clones were sequenced. Sequences were aligned m~nll~lly and are shown in Table 2.
B. RNA Sequences From the sixth and eleventh rounds, respectively, 27 of 29 and 21 of 35 sequenced ligands were unique. The number before the "." in the ligand name inflic~t~s 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 isshown in lower case letters. By definition, each clone includes both the evolvedsequence 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, (n), 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 f~milie~ in response to increased selective pressure (Table 2). Family 3, predominant (11/29 ligands) inround 6, has nearly disappeared (2135) by round 11. Sirnilarly, minor families 6 WO 96,~070~ PCT/U~ )5''~

5 through 9 virtually disappear. In contrast, only one (family l) of round eleven's predominant families ( 1, 2, 4 and 5) was detecte-l in round six. The appearance and disa~ea~ ce of f~milies roughly correlates with their binding ~ffiniti~os ,~lignm~-nt (Table 2) defines consensus sequences for f~miliPs 14 and 6-9 - (SEQ ID NOS: 56-63). The conC~nc-lc sequences of families 1-3 are long (20, 16 10 and 16, respectively) and very highly conselved. The concenc-ls sequences of families 1 and 2 contain two sequences in common: the trinucleotide TCG and the pent~nl-~leotide ACGAA. A related tetranucleotide, AACG, occurs in family 3. Thevariation 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 15 region.
The consensus sequences of family l and 2 are flanked by comrhon~nt~ry se,l~,r.-ces 5 or more nucleotides in length. These complementary sequences are not conserved and the mayority include minor disco"~ s. Family 3 also exhibits fl~nking complementary sequences, but these are more variable in length and 20 structure and utilize two nucleotide pairs of conserved sequence.
Confi~ence in the family 4 co~.sellsus sequence (Table 2) is limited by the small ~ be~ of lig~n~ls, the variability of spacing and the high G content. The pent~nucleotide, RCTGG, also occurs in f~Tnilies 5 and 8. T ig~n-ls of family 5 show other sequen,ce ~imil~rities to those of family 4, especially to ligand 11.28.
C. ~ffiniti~s The dissociation constants for representative m~mbers of families 1-9 and orphan ligands were deterrnined by nitrocellulose filter binding e~pel;ll~ents and are listed in Table 3. These calculations assume one RNA ligand binding site per WGA30 monomer. At the highest WGA concentration tested (36 ~lM 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 35 dissociation constants is 1.4 nM to 840 nM. Third, the binding for a number of ligan~l~, 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 ~lM. Fourth, on average eleventh round isolates have higher affinity than those from the sixth round. Fifth, the SELEX probably was not taken 40 to completion; the best ligand (11.20)(SEQ ID NO: 40) is not the dominant species.
Since the SELEX was arbitrarily stopped at the l 1th round, it is not clear that 11.20 WO 96/40703 PCT/U' 5~ S ~5 5 would be the llltim~P. 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 oligos,.~ch~rides (ie., the affinity of 11.20 is 5x105 greater than that of GlcNAc, Kd = 760 IlM, and 850 better than that of (GlcNAc)3.
Kd = 12 ~lM; Y.Nagata and M.Burger, 1974, supra). This observation validates the10 p.oposilion that coll~clitive elution allows the isolation of oligonucleotide ligands with ~ffinities that are several orders of m~gnitllde greater than that of the co~ ethlg sugar.
In addition these data show that even under conditions of high target density, 116 pmol WGA dimer/~l of beads, it is possible to overcome avidity problems and 15 recover ligands with nanomolar ~ffiniti~s. From the sixth to the eleventh round (Table 2), in response to increased selective pressure as int~ te~l by the hll~lov~llRllt in buLk Kd (Table 1), sequence famili~s with lower than average affinity (Table 3) are elimin~t~ 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, 40, and 19) for GlcNAc binding lectins from Ulex europaeus, Datura stramonium and Canavalia ensiformis were de~r~ by nitrocellulose partitioning. The results of this ~terrnin~tion 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. ~ lollium compares to a 3 to 10 fold difference in their affinity for oligomers of GlcNAc and validates the proposition that colllpeti~i~e 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.
40 Furthermore, if the oligonucleotide ligands bind exclusively to carbohydrate bindins 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-WO9~';C703 PCT/U' ~'0~155 5 fold dilution series, were added to three sets of binding reactions that contained WGA and an a-32P labeled RNA ligand (6.8, 11.20 or 11.24 (SEQ ID NOS: 13, 40 andl9); [RNA] final = [WGA]final = 15 nM). After a 15 minute incubation at room tel~ ld~UlC, the reactions were filtered and processed as in standard binding - eA~C~ t.c 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 5). These data do not rule out the possibility that (GlcNAc)3 binds one or more sites that are not bound by these RNA lig~n-lc QIlA.~ Alivt;ly, these data fit a simple model of coll,~Li~ive inhibition (Table15 5) and give estimates of 8.4, 10.9 and 19.4 ~lM for the Kd of (GlcNAc)3. These estimAtes are in good agreement with 1iLecl_Lulc values (12 ,uM @ 4 C, Nagata and Burger, 1974, supra; 11 ,uM @ 10.8 C, Van Landschoot et al., 1977, Eur. J.
Biochem. 79:275-283; 50 IlM, M.Monsigny et al., 1979, Eur J. Biochem. 98:39-45). These data confLrm the ploposiLion that co~ cLili~e elution with a specific20 carbohydrate targets the lectin's carbohydrate binding site.
Exarnple 5 Inhibition of WGA Aa~llll;llAl;ll~ Activity At 0.5 ~LM, RNA ligands 6.8 and 11.20 (SEQ ID NO: 13 and 40) completely 25 inhibit WGA m~iAr~ ggltltinAtion of sheep erythrocytes (Table 6). Ligand 11.24 (SEQ ID NO: 19) is not as effective, showing only partial inhibition at 2 ~lM, the highest concentration tested (Table 6). (GlcNAc)3 and GlcNAc completely inhibit ag~,lutinAtion at higher concentrations, 8 ~LM and 800 ~lM, respectively, (Table 6;
Monsigny et al., supra). The inhibition of AgrllltinAtion varifies the proposition that 30 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 ~gglutin~tion is a function of multiple carbohydrate binding sites.
An Alttqm~tive intel~cta~ion for the inhibition of agglutination is that charge repulsion prevents negatively charged WGAIRNA complexes from binding 35 carbohydrates (a n~cessAry 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 (pI = 4) 40 succinylated WGA is as effective as native WGA (pI = 8.5) in aggl~ltin~ting erythrocytes (M.Monsigny et al., supra).

WO 9~ 7Av3 PCT/U~ /vS ~-c Example 6 Secondary Structure of High Affinitv WGA T .io~nrl5 In favorable inst~nres~ co~ dti~e 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 base10 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.
Colllp~ e analyses of both family 1 and 2 sequences each yield a hairpin 15 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 sterns of individual ligands vary in sequence, length and structure (i.e., a variety of bulges and internal loops are allowed; Table 2). Qualit~ively it is clear that the stems are v~lid~t~l by Watson/Crick covariation and that by the rules of collly~aLi~e 20 analysis the stems are not direcdy 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, t~heir ~ccignment to a single family is questionable. Both ligand 11.7, 25 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, 30 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 Li~ands to Human L-Selectin The e~ç~ i" ,~t~l procedures outlined in this Example were used to identify and char~terize the 2'-NH2 RNA ligands to human L-selectin in Examples 8-12.
40 E~c~ elltal Procedures wog~ 703 PCT/US96/09455 5 A) Materials LS-Rg is a chi, llrl ;C protein in which the extr~rç~ r domain of human L-selectin is joined to the Fc domain of a human G2 immnnoglobulin (Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg, PS-Rg and CD22,B-Rg are analogous - constructs of E-sçl~ctin, P-selectin and CD22~ joined to a human Gl immunoglobulin Fc domain (R.M. 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 ~ur~l1ased from Ph~rm~ Biotech. Anti-L-selectin monoclonal antibodies: SKl l 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 synth~ci7~d by Operon. All other reagents and ch~mi~lc were purchased from co~ ,ial sources. Unless otherwise intli~te-l, eA~. ;~ c utilized HSMC buffer (1 mM CaCl2, 1 mM MgCl2, 150 mM NaCl, 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 sequen~e of the synthetic DNA template for the LS-Rg SELEX was randomized at 40 positions. This variable region wac flanked by N7 5' and 3' fixed regions (40N7). 40N7 transcript has the sequence 5' 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' taatacg~rt~t~t~gggaggacgatgcgg 3' (SEQ ID NO: 65) N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66) The fixed regions include primer ~nn~ling sites for PCR and cDNA synthesis ac well as a concçncllc 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
polymerace. Klenow extension conditions: 3.5 nmols primer SN7, 1.4 nmols 40N7, lX Klenow Buffer, 0.4 mM each of dATP, dCTP, dGTP and dTTP in a reaction volume of 1 ml.

WO g~'~0703 PCI/US96/09455 For subsequent rounds, eluted RNA was the template for AMV reverse transcriptase m.~ ted synthesis of single-stranded cDNA. These single-stranded DNA molecules were converted into double-stranded transcription templ~t~s by PCRarnplification. PCR conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgCl2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of Taq DNA
polylllclase. Transcription reactions contained 0.5 rnM DNA template, 200 nM T7 RNA polymerase, 80 mM HEPES (pH 8.0), 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-32P 2'-OH ATP.
The strategy for partitioning LS-RglRNA complexes from unbound RNA is outlined in Tables 7a and 7b. First, the RNA pool was incllb~t,ocl 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 inrllb~tin~ tbe washed beads in HMSC buffer co~ S mM EDTA in place of divalent cations. The 5 mM
20 elution was followed by a non-specific 50 mM EDTA elution. LS-Rg was coupled to protein A sepharose beads according to the m~nllf~turer's instructions (Ph"rm~cj~ Biotech).
The 5 rnM EDTA elution is a variation of a specific site elution strategy.
Although it is not a priori as specific as elution by carbohydrate co~ eli~ion, it is a 25 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 forrn bonds with the lectin's bound Ca~ because the low EDTA concentration does not appreciably increase tbe 30 buffer's ionic strength and the conforrnation of C-type lectins is only subtly altered in the ~bsence of bound calcium (unpublished observations cited by K. Drick~mer,1993, Biochem. Soc. Trans. 21:456-459).
In the initial SELEX rounds, which were performed at 4 ~C, the density of immobilized LS-Rg was 16.7 pmols/~ll of Protein A Sepharose 4 Fast Flow beads.
35 In later rounds, tbe 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 CI able 7b). Wash and elution buffers were equilibrated to the relevant incubation elatule. Beginning with the fifth round, SELEX was often done at more than WO96/40703 PCT/U' ~

5 one LS-Rg density. In each branch, the eluted m~t~ri~l from only one LS-Rg density was carried forward.
Before each round, RNA was batch adsorbed to 100 ,ul of protein A
se~halose beads for 1 hour in a 2 ml siliconized column. Unbound RNA and RNA
eluted with minim~l washing (two volumes) were combined and used for SELEX
10 input m~tPri~l 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 ~1 HSMC/wash). Bound RNA was eluted as two fractions, first, bound RNA
was eluted by incllb~tin,, and washing columns with 5 mM EDTA in HSMC without 15 divalent cations, second, the ,e~ ;..;n" elutable RNA was removed by incubating andlor washing with 50 mM EDTA in HSMC without divalents. The percc~LIge 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 id~ntic~lly to the SELEX beads to ~l~t~- ",il~ background bintling All unadsorbed, wash and 20 eluted fractions were counted in a Berlrm~n LS6500 scintill~tion 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 ~cCipi~Lil~g with is~,u~anol/ethanol (1: 1, v/v), the RNA was reverse transcribed into cDNA by 25 AMV reverse transcriptase either 1) at 48 ~ C for 15 min~ltes and then 65 ~C for 15 minutes or 2) at 37 ~C and 48 ~C for 15 minlltes each, in 50 mM Tris-Cl pH (8.3), 60 mM N~l, 6 mM Mg(OAc)2, 10 mM Dl-r, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/~Ll AMV RT. Transcripts of the PCR product were used to initiate the next round of SELEX.
C) Nitrocellulose Filter Binding Assay As ~srrihed in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to ~let~rmine the affinity of RNA ligands for LS-Rgand for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 35 0.45,um pore size, Millipore) were placed on a vacuum manifold and washed with 2 ml of HSMC buffer under vacuum. Reaction mixtures, cont~inin, 32p labeled RNA pools and unlabeled LS-Rg, were incubated in HSMC for 10 - 20 min at 4 ~C, room temperature or 37 ~C, filtered, and then imm~ t~-ly washed with 4 ml HSMC
at the same ~ dLulc. The filters were air-dried and counted in a Beckman 40 LS6500 liquid scintill~tion counter without fluor.

W O ~"~C703 PCT/U~ J~ 5 S LS-Rg is a dimeric protein that is the e*)r~SSiOn product of a recombinant gene constructed by fusing the DNA sequen~e that encodes the extr~e~ r domains of human L-selectin to the DNA that encodes a human IgG2 Fc region. For affinity c~lc~ tions, we assume one RNA ligand binding site per LS-Rg monomer (two per dimer). The monomer conce~ Lion is defined as 2 times the LS-Rg dimer collce~ Lion. The eqnilibrium (licsoci~tion constant, Kd, for an RNA pool or specific ligand that binds ~"ono~h~c;r~lly is given by the equation Kd = [Pf~[Rf~/[RP]
where, [Rf~ = free RNA corlcçntration [Pf~ = free LS-Rg monomer concentration [RP]= concentration of RNA/LS-Rg comrle~r~s Kd = ~i~SOCi~tionCOl~ ,~Ult A rearrangement of this equation, in which the fraction of RNA bound at eqnilihrillm is e~ ssed as a function of the total conc~ ~ion of the re~rt~ntc~ was used to c~l~nl~te Kds of monophasic binding curves:
q = (PT + RT + Kd ~ ((PT + RT + Kd)2 - 4 PT RT)1t2 ) q = fraction of RNA bound [PT~ = 2 x (total LS-Rg concentration) [RT] = total RNA cQI~r~ ion Many ligands and evolved RNA pools yield biphasic binding curves. Biphasic binding can be desçribed as the binding of two affinity species that are not in eqnilibrinm Biphasic binding data were evaluated with the eqll~tion q = 2p*Rt+Kdl+Kd2-[(pt+xlRl+Kdl)2-4ptxlRt]ll2 -[(P*x2Rt+Kd2)2-4ptx2Rt] 1/2, where X1 and X2 are the mole fractions of affinity species Rl and R2 and Kdl andKd2 are the col-esponding dissociation conct~ntc KdS were tl. t~"~.in~d by leastsquare fitting KdS were dete~nin~d by least square fitting of the data points using the graphics program K~lei~vraph (Synergy Software, Reading, PA).

D) Cloning and Sequen~ing Sixth, thirteenth (RT) and fourteenth (4 ~C) round PCR products were re-amplified with primers which contain either a BamHI or a HinDm restriction endonllcle~ce recogrution site. Using these restriction sites, the DNA sequenceswere inserted directionally into the pUC9 vector. These recombinant pl~cmirlc were transformed into E. coli strain DHSa (Life Technologies, Gaithersburg, MD).

wo ~ 703 PCT/US96/09455 5 Plasmid DNA was prepared according to the ~lk~lin~ hydrolysis method (PERFECTprep, 5'-3', Boulder, CO). Approximately 150 clones were sequenced using the Sequenase protocol (Amersham, Arlington Heights, IL). The resl~ltin~
ligand sequences are shown in Table 8.

10 E) Cell Binding Studies The ability of evolved ligand pools and cloned ligands to bind to L-selectin ~lGsenlGd in the context of a cell surface was tested in e~r~ ntc with isolated human peripheral blood mononuclear cells (PBMCs). Whole blood, collected from normal vollmt~çrs, was anticoagulated with S rnM EDTA. Six millilitto.r..c of blood 15 were layered on a 6 ml Histopaque gradient in 15 ml polypropylene tube and centrifuged (700 g) at room tG~ er~ e for 30 mintttes. The mononuclear cell layer was colltoct~l, diluted in 10 ml of Ca~/Mg~-free DPBS (DPBS(-); Gibco 14190-029) and centrifuged ~225 g) for 10 ,,,,,,llleS at room ttlll~Gl~lule. Cell pellets from two gradients were combined, le~u~el1ded in 10 ml of DPBS(-) and lccGllL~iruged as rlesçrihecl above. These pellets were resuspended in 100 ~LI of SMHCK buffer supplementPd with 1% BSA. Cells were counted in a hemocylolllGtel, diluted to 2x107 cells/rnl in SMHCK/1% BSA and imme~lizttely added to binding assays. Cell viability was monitored by trypan blue exclusion.
For cell binding assays, a constant nurnber of cells were titrated with 25 increasing concentrations of radiolabeled ligand. The test ligands were serially diluted in DPBS(-)/1%BSA to 2-tirnes the desired final concentration a~ at~ly 10 minlttes before use. Equal volumes (25 111) of each ligand dilution and the cell suspension (2x107 cells/ml) were added to 0.65 rnl eppendorf tubes, gently vortexed and incubated on ice for 30 minlttes At 15 minlttes the tubes were 30 revortexed. The ligand/PBMC suspension was layered over 50 ~11 of ice cold phth~l~te oil (1:1 = dinonyl:dibutyl phth~l~te) and rnicrofuged (14,000 g) for 5minntçS at 4 ~C. Tubes were frozen in dry ice/ethanol, visible pellets al~ ated 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 colllpetilion with the L-selectin specific blocking monoclonal antibody, DREG-56, while saturability of binding was tested by competition with unlabeled RNA. ExpeTiment~l procedure and conditions were like those for PBMC binding expeTimentc, except that the radiolabeled RNA ligand (final concentration 5 nM) was added to serial dilutions of the competitor before mixing with PBMCs.

CA 02223275 l997-l2-02 W O g~ 7~ PCT/U~ g1~5 5 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 (C. Foxall et al., 1992, supra). For these assays, the wells of Corning (25801) 96 well microtiter plates were coated with 100 ng of a sialyl-LewisX/BSA conjugate, air dried 10 overnight, washed with 300 ,ul of PBS(-) and then blocked with 1% BSA in SHMCK for 60 min at room telllpel~ e. RNA ligands were inc~lbatP~l with LS-Rg in SHMCK/1% BSA at room t~lllpeld~ for 15 min. After removal of the blocking solution, 50 111 of LS-Rg (lOnM) or a LS-Rg (lOnM)/RNA ligand mix was added to the coated, blocked wells and inr~lbat~ at room tC;lll~G dlule for 60 minlltt s The 15 binding solution was removed, wells were washed with 300 ~1 of PBS(-) and then probed with HRP conjugated anti-human IgG, at room te~ elalule to ~ ltil~te LS-Rg bintling, After a 30 minute inrllb~tion at room telllpe~aLul~ in the dark with OPD
peroxidase substrate ~Sigma P9187), the extent of LS-Rg binding and percent inhibition was determined from the OD4so.
Example 8 2'-NH~ RNA Ligands to Human L-selectin A. SELEX
The~starting RNA pool for SELEX, randomized 40N7 (SEQ ID NO: 63), 25 contained approximately 1ol5 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 estim~t~d to be approximately 10 !lM. 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 fold30 excess over background (Table 7a). The 50 mM eluted RNA from rounds 1 and 2 were amplified for the input m~teri~l for rounds 2 ar.d 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 rnM EDTA increased from 0.5 % in the first round to 8.4 %
35 in round 5 (Table 7a). An additional increase in specifically eluted RNA from the 10 ,uM LS-Rg beads was not observed in round 6 (Table 7a). To increase the stringency of selection, the density of irnmobilized 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 40 characterictics WO 96/40703 PCT/U' ,G~ S 'Ç~

S Binding e~cpe.hllel.Ls with 6th round RNA revealed that the affinity of the evolving pool for L-selectin was telllpel~Lule sensitive. Beginning with round 7, the SELEX was branched; one branch was continued at 4 ~C (Table 7a) while the other was con~ ctecl at room tell~c;lature (Table 7b). Bulk sequencing of 6th, 13th (rm - temp) and 14th (4 ~C) RNA pools revealed noticeable non-randomness at round six and dramatic non-randomess at the later rounds. The 6th round RNA bound monoph~cic~lly at 4 ~C with a dissociation constant of a~ploxilllately 40 nM, while the 13th and 14th round RNAs bound biph~ lly with high affinity Kds of approximately 700 pM. Tne molar fraction of the two pools that bound with high affinity were 24 % and 65 %, respectively. Tne 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++
/Mg++, plus 2 mM EDTA).
To monitor the progress of SELEX, ligands were cloned and ~equçnced from rounds 6, 13 (rm temp) and 14 (4 ~C). Sequences were aligned m~nll~lly and 20 with the aid of a co~ ul~r program that det~rrnin~s consensus sequences from frequently occl~rring local ~ t~

B. Sequences In Table 8, ligand sequences are shown in standard single letter code 25 (Cornish-Bowden, 1985 NAR 13: 3021-3030). The letter/number combination before the "." in the ligand name in~1ie~tes 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 in~hl(les both the evolved sequence and the associated fixed region, unless specifically stated 30 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, (n), following the ligand isolate number. These clones fall into thirteen 35 sequence families (I - XIII) and a group of unrelated sequences (Orphans)(SEQ ID
NOs: 67-117).
Two families, I and m are defined by ligands from multiple lineages. Both f~mili~s occur frequently in round 6, but only one farnily m ligand was identified in the final rounds. Six families (IV, V, VI, VII, vm, and possibly II) are each 40 defined by just two lineages which lirnits confidence in their consensus sequences.

CA 0222327=, 1997-12-02 wog6~c703 PCT~S~,~'o'~,tS~, 5 Five families (IX through Xm) are defined by a single lineage which precludes detçrmin~tion of concen~llc sequences.
T.ig~ntls 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 10 ligand's information content is extremely high and was therefore represented by a unique species in the starting pool. Family II ligands were not d~tec~d in the sixth round which is concict~nt with a low frequency in the initial population. An alternative explanation is sampling error. Note that a sequence of questionable relationship was ~etect~d in the sixth round.
The best defined conc~ncuc sequences are those of family I, AUGUGUA
(SEQ ~ NO: 118), and of family m, AACAUGAAGUA(SEQ ~ NO: 120), as shown in Table 8. Family m has two additional, variably spaced sequences, AGUC and ARWAG, that may be conserved. The tetranucleotide AUGWis found in the concçncuc sequence of families I, m, and VII and in families II, vmand IX. If this sequence is ci~nifi~nt, it suggests that the conserved sequences of ligands of family vm are circularly p~ rA The sequence AGAAiS found in the consensus sequence of f~miliçs IV and VI and in f~mili~s X and XIII.

C. ~ffiniti~5 The dissociation constants for representative ligands from rounds 13 and 14, in~ lin" all orphans, were tlçtçrmine~ by nitrocellulose filter binding expçrim~nts 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 dçterminPd but is estim~t~l to be approximately 10 ~M.
In general, ligands bind monophasically with dissociation constants ranging from 50 pM to 15 nM at 4 ~C. 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 ~C and room tenl~eld~llre, in general the ~ffinities decrease with increasing telllyel~ 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.

CA 0222327~ 1997-12-02 W O 96/40703 PCT/U'lCJ~5~'5 Example 9 Specificity of 2'-NH~ RNA Ligands to L-Selectin The affinity of L-selectin ligands to ES-Rg, PS-Rg and CD22~-Rg were ~et.orminç~ 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 103-fold lower and that for PS-Rg is about 104-fold less than for LS-Rg. Binding above background is not observed for CD22~-Rg at the highest protein concentration tested (660 nM), in~ aring that ligands do not bind the Fcdomain of the chim.oric constructs nor do they have affinity for the sialic acid binding site of an ~"nrelated lectin. The specificity of oligonucleotide ligand binding contrasts sha~rply with the binding of cognate carbolly~dtes by the selectins and co~ . the oposiLion that SELEX ligands will have greater specificity than carbohydrate lig~n-lc Example 10 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 d~mc-n~trate that they bind L-selectin presented in the context of a cell surface. Comparison of 2nd and 9th round RNAs (Figure 2) showsthat the evolved (9th round) ligand pool binds isolated PBMCs with high affinityand, as expected for specific binding, in a saturable fashion. The binding of round 2 RNA appears to be non-saturable as is char~cteri~tic of non-specific hin-ling 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 3). The saturability of binding is confirrn~cl by the data in Figure 4; > 90% of 5 nM 32P-labeled F14.12 RNA binding is competed by excess cold RNA. Specificity is demonstrated by tne results in Figure 5; binding of 5 nM
32P-labeled F14.12 RNA is completely competed by the anti-L-selectin blocking monoclonal antibody, DREG-56, but is unaffected by an isotype-m~tch~ d 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/USg~ 7 ''' Example 11 Inhibition of Binding to Sialyl-LewisX
Oligonucleotide lig,an~s, eluted by 2-5 rnM 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-LewisX 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 ICso of about 10 nM (Figure 6). Complete inhibition is observed at 50 nM F14.12. The observed inhibition is reasonable under the nt~l conditions; the Kd of F14.12 at room ~ d~ e 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 lowconcentrations of EDTA speçifi~ y elute ligands that bind the lectin dornain's carbohydrate binding site.

Example 12 Secondary Structure of High Affinity 2'- NH~ Ligands to L-Selectin In favorable inct~ es" colllp~LId~i~re 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 basepairing 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.
Colll~ala~ive analysis of the family I ~lignment suggests a hairpin structure inwhich 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 char~ten7ed ligands (Table 9).
The proposed structure for farnily m is also a hairpin with the conserved sequence, AACAUGAAGUA, contained within a variable length loop (Figure 7b).

WO 9f '~703 PCT/U~, ''.~S 1'5 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 ~lignm~nt 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 co~ aLible with the major variant sequences.

Example 13 ssDNA Ligands to Human L-Selectin The experim~t~l procedures outlined in this Example were used to identify and characterize ssDNA ligands to human L-selectin as described in Examples 14-21.
Experim~nt~l Procedures A) Materials Unless otherwise inllic~t~-l all m~t~n~l~ used in the ssDNA SELEX against the L-selectin/IgG2 chi~ .d, LS-Rg, were id~ntir~l to those of Example 7, paragraph A. The buffer for SELEX experim~ntc was 1 mM CaC12~ 1 mM MgC12.
100 mM NaCl, 10.0 mM HEPES, pH 7.4. The buffer for all binding affinity Lj differed from the above in cont~ining 125 mM NaCl, 5 mM KCl, and 20 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 identic~l to that described in Example 7, paragraph B except as noted below. Thenucleotide 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' fL~ed regions. The random DNA template was termed 40BH (SEQ ID NO: 126) and had the following sequence: 5'-ctacctacgatctgactagc<40N>gctt~ctctcatgtagttcc-3'. Theprimers 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 fLxed regions include primer annealing sites for PCR
~rnplification. The initial DNA pool contained 500 pmols of each of two types ofsingle-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 KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM

CA 0222327~ 1997-12-02 WO ~ 07û3 PCT/US9''~5 ''5 5 MgC12, 1 mM of each dATP, dCTP, dGTP, and dTTP and 25 U/ml of the Stoffel fragment of Taq DNA polymerase. After PCR arnplification, double stranded DNAs were end-labeled using ~32P-ATP. Complementary strands were separated by electrophoresis through an 8% polyacrylamide/7M urea gel. Strand separation results from the molecular weight dirr~l~nce of the strands due to biotintylation of 10 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 ~lc~ Lion.
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 1 5 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 deLe- " ~ e the affnity of 20 ssDNA ligands for LS-Rg and for other proteins. For these exp~rimlont~ a Gibco BRL 96 well manifold was substituted for the 12 well Millipore manifold used in Example 7 and radioactivity was ~let~rrnined with a Fujix BAS100 phosphorimager.Binding data were analyzed as described in Exarnple 7, paragraph C.

25 D) Cloning and Sequencing Thirteenth, fifteenth and seventeenth round PCR products were re-arnplified with primers which contain either a BamHI or a HinDm restriction endonuclease recognition site. Approximately 140 ligands were cloned and sequenced using the procedures described in Example 7, paragraph D. The resulting sequences are 30 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 35 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 40 histoplaque by standard techniques. Cells (500 cellslrnL) were incubated with CA 0222327~ 1997-12-02 wo ~ 0703 Pcr/uss6/094s5 5 fluorescein labeled oligonucleotide in 0.25 mL SMHCK buffer (140 mM NaCl, l rnM MgCl2, 1 mM CaCl2, 5 mM, KCl, 20 mM HEPES pH 7.4, 8.9 mM NaOH, 0.1% (w/v) BSA, 0.1% (w/v) sodium azide) at room temperature for 15 minlltes Fluorescent staining of cells was quantified on a FACSCaliber fluorescent activated cell sorter (Becton Dickinson, San Jose, CA).
To ~x~minP the ability of oligonucleotides to bind leukocytes in whole blood, 25 ,ul aliquots of hep~rinice~ whole blood were stained for 30 min at 22~ C with 2 ~lg CySPE labeled anti-CD45 (generous gift of Ken Davis, Becton-Dickinson) and 0.15 ~LM ~'l'l'C-LD201Tl (synth~-si7ed with a 5'-Fluorescein phosphoramidite by Operon Technologies, ~l~m~ CA; SEQ ID NO: 185). To (letermin~ specificity, 15 other samples were stained with FITC-LD201Tl in the presence of 0.3 ,uM DREG-56 or 7 ,uM unlabeled LD201Tl; or cells were reassayed after addition of 4 mM
EDTA. The final concentration of whole blood was at least 70% (v/v). Stained, concentrated whole blood was diluted 1/15 in 140 mM NaCl, 5 mM KCl, 1 mM
MgC12, 1 mM CaC12, 20 mM HEPES pH 7.4, 0.1% bovine serum albumin and 20 0.1% NaN3 im m~ tely prior to flow cytometry on a Becton-Dickinson FACS
Calibur. Lymphocytes and granulocytes were gated using side scatter and CD45CyPE staining.

F) Synthesis and Chara~t~ri7~tion of Multimeric Oligonucleotide T .ig~ntlc Synthesis of Branched Dimeric Oligonucleotide Complexes Dimeric oligonucleotides were synthesized by standard solid state processes, with initiation from a 3'-3' Syrnmetric T inking CPG (Operon, ~l~mto~ CA).
Branched complexes contain two copies of a truncated L-selectin DNA ligand, eachof which is linked by the 3' end to the above CPG via a five unit ethylene glycol 30 spacer (Figure 8A). Each ligand is labeled with a fluorescein phosphoramidite at the 5' end (Glen Research, Sterling, VA). Branched dimers were made for 3 truncates of LD20 lT 1 (SEQ ID NO: 142). The truncated ligands used were LD201T4 (SEQ
ID NO: 187), LD201T10 (SEQ ID NO: 187) and LD201Tl (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 tetrarneric protein, each subunit of which has a biotin binding site. 5' and 3' biotintylated DNAs were syntht~.ci7~d by Operon Technologies, Inc (~l~mtorl~ CA) CA 0222327~ 1997-12-02 WO 9~'~C i~3 PCT/US96. 0~ '-5 5 using BioTEG and BioTEG CPG (Glen Research, Sterling, VA), respectively. The expected stoichiometry is 2 to 4 DNA molecules per complex. SAlbio-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 LD201Tl (SEQ ID NO: 185). The bio-DNA/SA multivalent complexes were 10 generated by inc-lb~tin, biotin modified oligonucleotide (1 mM) and fluoroscein labeled streptavidin (0.17 mM) in 150 mM NaCl, 20 rnM HEPES pH 7.4 at room telllpeld~ c; for at least 2 hours. Oligonucleotide-streptavidin complexes were used directly from the reaction mixture wit_out additional pllrific~tion of the Complex from free streptavidin or oligonucleotide.
Synthesis of a Dumbell Dimer Multivalent Complex A "dllml~ll" DNA dimer complex was form~ tP~ from a homobifunctional N-hydroxysuccinimidyl (or NHS) active ester of polyethPlenP glycol, PEG 3400 MW, and a 29mer DNA oligonucleotide, NX303 (SEQ ID NO: 196), having a 5' 20 termin~l Amino Modifier C6 dT (Glen Research) and a 3~-3~ tPrmin~l 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 oligonucleo~ide by reverse phase chromatography. The dimer was then purified 25 from the monomer by anion exchange HPLC. The oligonucleotide was labeled at the 5' terminuc with fluorescein as previously described.

Synthesis of a Fork Dimer Multivalent Complex To synthPci7P the fork dimer multivalent complex (Figure 8D), a glycerol 30 was ~tt~hP~l by its 2-position to one t~rrninll5 of a linear PEG molecule (MW 20 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' ter T inllc of the truncated DNA nucleic acid ligand NX303 (SEQ ID NO: 196). The conjugation reaction was in DMSO
35 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 gC,~lG iO3 PCT/US9G~9 1'5 S Characteri_ation of ~llltim~ric Oligonucleotide T .ig~n~l~
The binding of dimeric and m1lltim~ric oligonucleotide complexes to human peripheral blood monon--cl~r cells was analyzed by flow cytometry as described in Exarnple 13, paragraph D.

10 G) Photo-Crosslinking A photo-cros.clinkin~ version of DNA ligand LD201T4 (SEQ ID NO: 187) was synth~si7~1 by replacing nucleotide T15 (Figure 12) with 5-bromo-deoxyuracil.
4 nmol of 32P-labeled DNA was incubated with 4 nmol L-selectin-Rg in 4 ml lX
SHMCK + 0.01 % human serurn albumin (w/v), then irradiated at ambient 15 te~ LuLe with 12,500 pulses from an excimer laser at a fii~t~n~e of 50 cm and at 175 rnJ/pulse. Protein and DNA were precipitated with 400 ~Ll 3 M sodium acetateand 8.4 ml ethanol followed by incubation at -70 degrees C. F'le~i~itated m~tt ri~l was centrifuged, vacuum dried and resuspended in 100 1l1 0.1 M Tris pH 8.0, 10 mM CaC12. Fourty-five ~lg chymoLLy~hl were added and after 20 min at 37 20 degrees C, the m~t~.ri~l was loaded onto an 8% polyacrylamide/7 M urea/ lXIBEgel and ele~ ~holesed until the xylene cyanole had migrated 15 cm. The gel was soaked for 5 min in lX TBE and then blotted for 30 min at 200 mAmp in lXTBE
onto Immobilon-P (Millipore). The membrane was washed for 2 min in water, air dried, and an autoradiograph taken. A labeled barld running slower than the free25 DNA band, represellting a chymotryptic peptide crosclink~cl 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 crosclink~d amino acid, F82 of the lectin domain.
H) Lymphocyte Tr~ffickin,, Experiments Human PBMC were purified from heparinised blood by a Ficoll-Hypaque gradient, washed twice with HBSS (calcium/magnesium free) and labeled with 51Cr (~m~r~h~m). After labelina, the cells were washed twice with HBSS (cont~inin,, 3~ calcium and m~gn~sillm) and 1 % bovine semm albumin (Sigma). Female SCID
mice (6-12 weeks of age) were injected intravenously with 2X106 cells. The cellswere 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 rnice40 were euth~ni.ced PLN, MLN, Peyer's patches, spleen, liver, lungs, thymus, CA 02223275 l997-l2-02 W O g~"~703 PCTAJS9GJ~ c 5 kidneys and bone marrow were removed and the counts incorporated into the organs del~l "~ r1 by a Packard gamma counter. In a second protocol, 2x106 human PBMC, purified, labeled, and washed as described above, were injected intravenously into female SC~) mice without antibody or oligonucleotide pretre~trn~t One to 5 min prior to injecting the cells, the animals were injected with 10 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 initi~t~o~l by coupling to a dT-5'-CE polyslylelle support (Glen Research), resliltin~ in a 3'-3' terminal phosphodiester linkage, and having a 15 5' t~nin~l an Amino Modifier C6 dT (Glen Research). Once NX288 and NX303 were synth~osi7eA, a 20,000 MW PEG2-NHS ester (Shearwater Polymers, Huntsville, AL) was then coupled to the oligonucleotide through the 5' arnine moiety. The molar ratio, PEG:oligo, in the reactions was from 3:1 to 10:1. The reactions were performed in 80:20 (v:v) 100 rnM borate buffer pH 8: DMF at 37~ C20 for one hour.

I) Inhibition of L-selectin Binding to Sialyl LewisX
SLeX-BSA (Oxford GlycoSystems, Oxford, UK) in lX PBS, without CaC12 and MgC12; (GIBCO/BRL) was immobilized at 100 ng/well onto a microtiter plate by25 overnight incubation at 22~ C. The wells were blocked for 1 h with the assay buffer consisting of 20 mM HEPES, 111 mM NaCl, 1 mM CaC12, 1 mM MgC12, 5 mM KCl, 8.9 rnM NaOH, final pH 8, and 1% globulin-free BSA (Sigma). The reaction mixtures, incubated for 90 min with orbital .~h~kin," contained 5 nM L-Selectin-Rg, a 1:100 dilution of anti-human IgG-peroxidase conjugate (Sigma), and 0 - 50 nM of competitor 30 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 O-phenylene~ rnine dihydrochloride peroxidase substrate (Sigma) by sh~king in the dark at 22~ C. Absorbance was read at 450 nm on a Bio-Kinetics Reader, Model EL3 12e (Bio-Tek Instruments, Laguna Hills, CA). Values shown represent the mean + s.e from 35 duplicate, or triplicate, samples from one representative experiment.

W O 9C~/~c~3 PCT/U~r~/~5 Example 14 ssDNA T i~n~ls 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 10 constant of randomized ssDNA to LS-Rg is estim~tt rl to be appl~kill.atloly 10 ~M.
The SELEX protocol is outlined in Table 11.
The initial round of SELEX was ~e~rulllRd at 4 ~C with an LS-Rg density of 16.7 pmol/~Ll of protein A sepharose beads. Subsequent rounds were at room t~lllL~c;ldLUlc~ except as noted in Table l l. The 2 mM EDTA elution was omitted from 15 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 ~mplifi~d forthe input m~tt~ lc 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 m~t~ri~l 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 ~C, the dissociation constant of round 7 ssDNA was 60 nM.
Binding ~L~lilllents with 7th round DNA revealed that the affinity of the evolving pool for L-selectin was weakly temperature sensitive (Kds: 60 nM, 94 nMand 230 nM at 4 ~C, room telllpeld~ and 37 ~C, respectively). To ~nh~nre the selection of ligands that bind at physiological temperature, rounds 8, 13, 16 and 17 were performed at 37 ~C. Although temperature sensitive, the affinity of round 15 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 5 and dramatic non-randomness at round 13. T igan~s were cloned and sequenced from rounds 13, 15, and 17. Sequences were aligned m~nu~lly and with the aid of a NeXstar computer program that ~etermines consensus sequences from frequently occurring local ~lig,nm~nts.

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 PCT/US9''0~ 1~5 5 By definition, each clone includes both the evolved sequence and the associated fixed region, unless specifically stated othenvise A unique sequence is operationally defined as one that differs from aIl others by three or more nucleotides.
Sequ~n~es that were isolated more tha~n once are inl1jr~t~d by the par~nth~oti~lnumber, (n), following the ligand isoIate number. These clones fall into six families 10 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 15 complelllelltaly sequences that are 3 to 5 nucleotides in length. The stati~tit~l rlcJ~ re of family 1 suggests that the ~l~e,Lies of the bulk population are a reflection of those of f~mily 1 ligands. Note that ssDNA family I and 2'-NH2 family I share a com~mon sequence, CAAGGCG and CAAGGYG, respectively.
Family 2 is represented by a single sequence and is related to f~mily 1. The 20 ligand contains the absolutely conserved CAAGG and highly conserved ACG of family 1 although the spacing between the two elem~-nt~ is strikingly dirr~lcl~t (23 co~llyalc;d to 6 nucleotides).
F~rnilies 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 ~"~ d C. ~ffinitiçs 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 detenninç~l but is estimated to be approximately 10 ~LM.
At room tel~lpeldlu.e, the dissociation constants range from 43 pM to 1.8 nM
which is at least a 5x103 to 2x105 fold improvement over randomized ssDNA
(Table 13). At 37 ~C, the Kds range from 130 pM to 23 nM. The extent of t~ eldLule sensitivity varies from insensitive (ligands LD 122 and LD127 (SEQ IDNO: 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 LD733 CA 0222327~ l997-l2-02 W O 9~/~C703 PCT/U'~'O~rS

5 (SEQ ID NOS: 133, 134, 132, and 146)), the difference in affinity at room t~lllpc:la~ e and 37~C is about 4-fold.
The observed ~ffinities of the evolved ssDNA ligand pools reaffirm our proposition that it is possible to isolate oligonucleotide ligands with ~ffinities that are several orders of m~gnitll~le greater than that of carbohydrate ligands.
Example 15 Specificity of ssDNA Ligands to L-Selectin The affinity of lc~lcsentative cloned ligands for LS-Rg, ES-Rg, PS-Rg, CD22~-Rg and WGA was ~letpnnine~l by nitrocellulose partitioning and the results 15 shown in Table 14. The ligands are highly specific for L-selectin. The affinity for ES-Rg is about 103-fold lower and that for PS-Rg is about 5x103-fold less than for LS-Rg. Binding above background is not observed for CD22,B-Rg or for WGA at 0.7 and 1.4 ~LM protein, respectively, intli~ting that ligands neither bind the Fc domain of the chimeric constructs nor have affinity for unrelated sialic acid binding 20 sites.
The specificity of oligonucleotide ligand binding col~ sl~, 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 9shows that the round i5 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 32P-labeled round 15 ssDNA is completely competed ~ 35 by the anti-L-selectin blocking monoclonal antibody, DREG-56, but is unaffected by an isotype-matched irrelevant antibody. In analogous experimPntc, LD201Tl (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 feasibiIity of using immobilized, purified protein to isolate ligands against a cell surface protein and demonstrate the specific binding of Wo ~ 0703 Pcr/uss6/094s5 5 the round 15 ssDNA pool and of ligand LD201Tl to L-selectin in the context of a cell surface.
The binding of LD201T1 to leukocytes in whole blood was e~mintorl by flow cytometry. Fluorescein isothiocyanate (FTlC)-conjugated LD201T1 specifically bind human lymphocytes and neutrophils (Figure 1 lA/B); binding is 10 inhibited by competition with DREG-56, unlabeled LD201, and by the addition of 4 mM EDTA (Figure 1 lA/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 inct~n~es, colll~a,d~iv~ analysis of aligned sequences allows tle~ ctiQn of secondary structure and structure-function relationships. If the nucleotides at two positions in a seqlle~e covary according to Watson-Crick basepairing rules, then the' nucleotides at these positions are apt to be paired.
20 Nonconserved sequences, espe~i~lly those that vary in length are not apt to be directly involved in function, while highly conserved sequence are likely to be directly involved.
Colllp~Live analysis of 24 sequences from family 1 strongly ~,u~po-L~, a hairpin secondary structure for these ligands (Figure 12). In the figure, consensus 25 nucleotides are specified, with invariant nucleotides in boid 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 ~e~uce-l 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 asymmetric ~l, AA internal 30 loop or "bulge." Two of the three base pairs in the upper stem and 6 of 7 in the lower stem are v~lid~te~l 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 T15) in binding the target protein.
The site of oligonucleotide binding on L-selectin can be de~l~ce~ from a set of colllpeLiLion ex~elimellts. DREG56 is an anti-L-selectin, adhesion blocking monoclonal antibody that is known to bind to the lectin domain. Binding of threeunrelated 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-m~tch~ control. In cross-competition experimt ntc, LD201T1, LDl74Tl, or LD196T1 prevented radio-labeled LD201Tl from binding to LS-Rg, consistent CA 02223275 l997-l2-02 W O 96/40703 PCT/U'~J~g1~5 5 with the premise t'nat the ligands bind the same or overlapping sites. The blocking and co~ ion e~elillRI1ts, taken together witn divalent cation-dependence of binding, suggest that all three ligands bind to the lectin dom~in. This conclusion has been verified for LD201 by photo-crosslinkin, e}~, ;~ tc If T15 of LD201T4 (SEQ ID NO: 187; Figure 12) is replaced with 5-bromo-10 uracil, the resulting DNA photo-crosclinks at nigh yield (17%) to LS-Rg following irradiation with an excimer laser as described in Example 13, paragraph G. The nigh yield of crosclinking inAit~tec a point contact between the protein and T15.
Sequencing of the ch~llwLIy~Lic peptide corresponding to this point contact revealed a peptide deriving from the lectin domain, F82 is tne crosslinking, amino acid. Thus, 15 F82 col~t~c~i T15 in a st~king arrangement that perrnits high yield photo-crosclinking By analogy to the ~ clule of tne 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 hinAerin,a, access to the carbohydrate binding site or in altering the collf~,lll~ion of the lectin domain upon DNA binding.

Example 18 L-Selectin ssDNA Ligand Truncate Data Initial e~ tc to define the minim~l high affinity sequence of family 1 ligands show that more than the 26 nucleotide hairpin (Figure 12; Table 13) is required. ~ ig~nAs corresponding to the hairpin, LD201T4 (SEQ ID NO: 187) and LD227Tl (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 progel~iLols. The affinity of LD201T3 (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 LD201Tl (SEQ ID NO: 185) is not cignific~ntly altered (Tables 12 and 13).
Additional e~rt~rimentc show that truncates LD201T10 (SEQ ID NO: 188) and LD227Xl (SEQ ID NO: 191) bind with ~ffiniti~s 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 W 096/40703 PCT/U~ 7'~

S sequences. Fu-Lhellllore, LD201's bulge is at the 5'-end of the original stem base while that of LD227 is at the 3'-end. Thus, tne two ligands do not present an obvious concenc-ls structure. Removal of the loop (LD201) or scrambling or trllnr~ting the sequence (LD227) rliminichçs affinity, suggesting that the bulged sequences may be directly involved in binding. Note that although LD201T3 is 10 longer than LD201T10, it is unable to forrn the single stranded loop and extended stem because of the position of the trlmc~t~d ends.

Exarnple 19 Inhibition of Binding to Sialyl LewisX
Sialyl Lewis~ is the minim~l carbohydrate ligand bound by selectins. The ability of ssDNA ligands to inhibit the binding of L-selectin to Sialyl LewisX was d~t~ in colll~ti~ion ELISA assays as described in Example 13, paragraph I.
LD201Tl (SEQ ID N~O: 185 ), LDl;'4Tl (SEQ ID NO: 194) and LD196Tl (SEQ ID
NO: 195) inhibited LS-Rg binding to irnmobilized SLeX in a dose dependent manner20 with ICsos of ~ro~ill,~tely 3 nM. This is a 105-106-fold improvement over thepublished ICso values for SLeX 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 25 lectin domain's carbohydrate binding site.

Example 20 Inhibition Of Lymphocyte Tr~fficking by L-Selectin ssDNA Ligands Lymphocyte tr~ffickin,, to peripheral lymph nodes is exquisitely dependent 30 on L-selectin. Since the ssDNA ligands binds to human but not rodent L-selectin, a xenogeneic lymphocyte tr~fficking system was established to evaluate in vivo efficacy. Human PBMC, labeled with 51Cr, were injected intravenously into SCID
mice. Cell tr~fficking was detçn nine~l 1 hour later. In this system, human cells traffic to peripheral and m~Sentçric lymph nodes (PLN and MLN). This 35 ~rCum~ tion is inhibited by DREG-56 (Figure 13) but not MEL-14, a monoclonal antibody that blocks murine L-selectin-dependent trafficking. In initial expçnm~rltc cells were incubated with either DREG-56 or 3' capped and PEG-modified oligonucleotide before injection. NX288 (SEQ ID NO: 193) inhibited tr~fficking of cells to PLN (Figure 13) and MLN in a dose-dependent fashion but had no effect on 40 the accumulation of cells in other organs. At the highest dose tested (4 nmol), W 0 9~'~C7~3 PCT/U',~~1~r~'5 inhibition by the DNA ligand was comparable to that of DREG-56 (13 pmol), while a scrambled sequence had no ~ignifi~nt effect (Figure 13). The activity of LD174Tl (SEQ ID NO: 194) was similar to that of NX288.
To d~tt rminç if the modified oligonucleotide was effective when it was not pre-in~nb~t~cl 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 tr~fficking to PLN and MLN while the scrarnbled sequence had no effect (Figure 14). Therefore, the modified oligonucleotide did not require pre-incubation with the cells to effectively block tr~ffi~L ing These expe, ;",~"~ ml~n~trate~ in vivo, the efficacy of oligonucleotide ligands in inhibiting a L-selectin depçndçnt process.

Example 21 L-Selectin Nucleic Acid Li~and Multimers Multivalent Compl~Y~s were made in which two nucleic acid ligands to L-selectin were conjugated together. Multivalent Comp1to~s 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 inten~efl to increase the binding energy to f~cilit~t~ better binding ~ffiniti~s through slower off-rates of the nucleic acid li~nds These multivalent Complexes may be useful at lower doses than their monomeric counterparts. In addition, high molecular weight (20kD) polyethylene gylcol (PEG) was in~ d.ocl in some of the Complexes to decrease the in vivo clearance rate of the complexes. Specifically, the nucleic acid ligands incol~u.aLed 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 e~t~rnin~d in flow cytometry. The multivalent Complexes exhibited sirnilar - specificity to the monomeric forms, but erlh~nce-l affinity as well as improved (i.e., slower) off-rate for human lymphocytes. Titration curves, obtained from incubating - fluorescently labeled monomeric FITC-LD201T1 with peflpheldl blood mononuclear cells (PBMC) purified human lymphocytes, in~lir~t~d that binding to cells is 40 saturable. Half-saturation fluorescence occurred at 3 nM oligonucleotide. In contrast, the branched dimeric ~l l C-LD201T l and bio-LD201T1/SA multivalent WO 9~ D70~ PCT/US~f/~S 1rS

5 Complexes exhibited half-saturation at approximately 0.15 nM, col,c~,~onding to an apparent 20-fold increa~se in affinity. In similar e~spe,;~ ntc, half saturation of the dumbell and fork dimers of LD20lT4 was observed at 0.1 and 0.6 nM, respectively, conl~alcd to 20 nM for monomeric LD201T4.
Kinetic coll~c~ion e~;lilll~ll~7 were ~elrolllled on monomeric nucleic acid 10 ligands and multivalent Complexes. Kinetic competition e~ ts 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 Comrlçxçs was ~çterminç~l by addition of 500 nM nnl,th.~l~od oligonucleotide to cells stained with fluorescently labeled ligand and mea~surement of 15 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 LD201Tl branched dimer and biotin conjugate multivalent Complexes exhibited a~a~ off-rates sevèral 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, d~termin~-l under the same con~lition~. A multivalent Complex cu~ g a non-binding nucleic acid sequence did not stain cells under id~ntic~l conditions and did not compete in the off-rate experim~nts The off-rate of the LD201T4 ~llmhelland fork dirners is faster than the LD201Tl branched dimer and is better than all monomers tested. These results confirm the proposition that dimeric and mllltim~c 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 e~e,; ~ nt~l 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.
Ex~e~ RllL~l Procedures A) Materials Unless otherwise indicated, all m~t~n~lc 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 experim~ntc, was 1 mM CaC12, 1 mM MgC12, 140 mM NaCl, 5 mM KCl, and 20 mM HEPES, pH 7.4. A soluble form of L-selectin, collc~ollding to the CA 02223275 l997-l2-02 W 0 96/40703 PCT/U'~I~91S5 5 extracellular dom~inc, was purchased from R&D Systems and used for some nitrocellulose filter binding e~ t!i.

B) SELEX
The SELEX procedure is described in detail in United States Patent 10 5,270,163 and elscwllclc. Procedures are essçnti~lly idçntic~l to those in Examples 7 and 13 except as noted. The variable regions of synthetic DNA tçmrl~tçs were r~nflomi7~d 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' t~t~g~ c~ nl~ggaggacgatgcgg 3' (SEQ ID NO: 65) N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66) The initial RNA pool was made by first Klenow e~t~n-lin~ 3 nmol of synthetic single stranded DNA and then transcribing the res lltin~ double stranded mnleclllçs with T7 RNA poly~ se. Klenow extension conditions: 6 nmols primer 20 5N7, 3 nmols 30N7 or 40n7, lX Klenow Buffer, 1.8 mM each of dATP, d~
dGTP and dTTP in a reaction volume of 0.5 ml.
For ~ullsey~lent rounds, eluted RNA was the temrl~tç for AMV reverse Lli."c~ n;.ce m~ tçd :jy~l~lesis of single-str~n-l~cl cDNA. These single-stranded DNA molecules were converted into double-stranded transcription templates by PCR25 ~mplific~tion. PCR conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgC12, 0.2 mM of each dATP, dCTP, dGTP, and dTTP, and 100 U/ml of Taq DNA polymerase. Tla.lscli~tion reactions contained one third of the purified PCRreaction, 200 nM T7 RNA polymerase, 80 mM HEPES (pH 8.0), 12 mM MgC12, 5 mM DTT, 2 mM spçnni-linç, 1 mM each of 2'-OH ATP, 2'-OH GTP, 3 mM each 30 of 2'-F CTP, 2'-F UTP, and 250 nM a-32P 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 esc~o-n~i~lly identical to that of Example 7, paragraph B.
In the initial SELEX rounds, which were performed at 37 ~C, the density of 35 irnmobilized LS-Rg was 10 pmols/~ll of Protein A Sepharose 4 Fast Flow beads.LS-Rg was coupled to protein A sepharose beads according to the manufacturer's instructions (Ph~nn~ Biotech). In later rounds, the density of LS-Rg was reduced (Table 15), as n~e~ l to increase the stringency of selection. At the seventh round, both SELEXes were branched. One branch was continued as 40 previously described (Example 7, paragraph B). In the second branch of both WO 96/40703 PCT/U~ 155 5 SELEXes, the RNA pool was pre-annealed to oligonucleotides that are complementary to the S' and 3' fixed sequences. These rounds are termed "counter-selected" rounds. Before each round, RNA was batch adsorbed to 100 ,ul of protein A sepharose beads for 15 minlltes in a 2 rnl siliconized colurnn. Unbound RNA and RNA eluted with minim~l washing (two volumes) were combined and used for 10 SELEX input material. For SELEX, extensively washed, immobilized LS-Rg was batch inrub~t~cl with pre-adsorbed RNA for 1 to 2 hours in a 2 rnl column with co~ rocking. Unbound RNA was removed by extensive batch washing (500 ~11 SHMCK 140/wash). In addition, the counter selected rounds were extensively washed with buffer cont~inin~ 200 nM of both complem~nt~ry oligos. Bound RNA
15 was eluted as two fractions; first, bound RNA was eluted by inrllb~ting and wasning columns with 100 ~lL S mM EDTA in SHMCK 140 without divalent cations; second, the rem~ining elutable RNA was removed by inrub~ting and/or washing with 500 ~L 50 mM EDTA in SE~CK 140 without divalents. The percentage of input RNA that was eluted is recorded in Table 22. In every round,20 an equal volume of protein A sepharose beads without LS-Rg was treated id~-ntir~lly to the SELEX beads to deterrnin~ background bin-lin~ All unadsorbed, wash and eluted fractions were counted in a Beckrnan LS6500 srintill~tion counter in order to monitor each round of SELEX.
The 5 rnM EDTA eluates were processed for use in the following round 25 (Table 15). After ~r~cipitating with isop~opallol/ethanol (1:1, v/v), the RNA was reverse transcribed into cDNA by AMV reverse transcriptase either at 48 ~C for 15 u~es and then 65 ~C for 15 minlltes in 50 rnM Tris-C1 pH (8.3), 60 rnM NaCl, 6 rnM Mg(OAc)2, 10 mM DTT, 200 pmol DNA primer, 0.5 mM each of dNTPs, and 0.4 unit/~lL AMV RT. Transcripts of the PCR product were used to initiate the next 30 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 35 and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 ~m pore size, Millipore) were placed on a vacuum manifold and washed with 3ml of SHMCK 140 buffer under vacuum. Reaction mixtures, cont~ining 32p labeled RNA pools and unlabeled LS-Rg, were incubated in SHMCK 140 for 10 -20 min at 37 ~C, and then immetli~tely washed with 3 ml SHMCK 140. The filters 40 were air-dried and counted in a Bec~m~n LS6500 liquid s~intill~tion counter without WO9''1~/03 PCT/U',~ 1~51J5 S fluor. Alternatively, binding studies employed 96 well micro-titer manifolds essentially as ~iesrril~e-l in Exarnple 13, paragraph E.

D) Cloning and Sequen~in, 12th round PCR products were re-amplified with primers which contain 10 either a BamHI or a HinDm restriction endonuclease recognition site. Using these restriction sites, the DNA sequences were inserted directionally into the pUC9 vector. These recc.lllbi~ t pl~cmi~ls were transformed into E. coli strain DH5a (Life Technologies, ~ithl . ~I)ulg, MD). Plasmid DNA was prepared according to the ~lk~lin~ lysis method (Quiagen, QIAwell, Chattsworth CA). Approximately 300 15 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 20 surface was tested by flow cytometry ek~, ;"~nts with human lymphocytes.
Briefly, peripheral blood monoml~le~r cells (PBMC) were purified on histoplaque by standard techniques. To evaluate leukocyte binding by unlabeled 2'-F ligands,cells (500 cells/mL) were in~ub~ted with fluorescein labeled FITC-LD201T1 (SEQ
ID NO: 185) in the presence of increasing concentrations of individual, unlabeled 2'-25 F ligands in 0.25 mL SMHCK buffer (140 mM NaCl, 1 rnM MgC12, 1 mM CaCl2,5 mM, KCl, 20 mM HEPES pH 7.4, 8.9 mM NaOH, 0.1% (w/v) BSA, 0.1%
(w/v) sodium azide) at room te~lyeldluLe for 15 minutes. Fluorescent st~ining ofcells was quantified on a FACSCaliber fluorescent activated cell sorter (Becton Dickinson, San Jose, CA). The affinity of the 2'-F competitor was c~ ted 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 1ol4 molecules (0.7 nmol RNA). The SELEX protocol is outlined in Table 15 and Example 22. All rounds were selected at 37~C. The dissociation constant of randomized RNA to LS-Rg is estim~t~d to be ayylu~ ately 10 ~I. ~fter six rounds the pool ~ffinities had 40 improved to approximately 300 nM. An aliquot of the RNA recovered from the seYenth round was used as the starting material for the first counter-selected rounds.

CA 0222327~ 1997-12-02 WO ~"'C70~ PCT/U~ v3 1~5 5 Five rounds of counter-selection and five additional standard rounds were pc;lr~llled in parallel. Thus, a total of twelve rounds were pelrolllled in both bldl~ches of both SELEXes: 30N7, counter-selected 30N7, 40N7 and counter-selected 40N7. The ~ffinities of each of the 12th round pools ranged from 60 to 400 pM. T i~n~ls were cloned from these pools.
B. Sequences of 2'-F RNA T.ig~nflc to L-Selectin In Table 16, ligand sequences are shown in standard single letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). Fixed region sequence is shown in lower case letters. By definition, each clone in~ludes both the evolved sequence and 15 the associated fLlced 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 icol~te~l more than once are in~ tPd by the p~ ulllbel~ (n), following the ligand isolate llulll~el.
The 30N7 and 40N7 SELEX final pools shared a coll" u~n major sequence 20 f~mily, even though ide~tir~l 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, RYGYGUWUCRAGY or RYGYGWWWUCRAGY. These motifs define f;~mily 1. Within the farnily there are three subf~miliP-s Subfamily la ligands (53/66) contain an additional sequence 25 motif, CUYARRY, one nucleotide 5' to the family 1 concencnc motifs. Subfamilylb (9/66 unique sequences) lacks the CUYARRY motif. Subfamily lc (5/66) is also miccing the CUYARRY motif, has an A inserted beLw~ll the Y and G of consensus YGW and lacks the concçnc-ls GA base pair. The ~ignific~nce of the sequence ~ubr~llliliP,s is reflected in the pos~ tP~l secondary ~l-u-;lule of the ligands (Example 30 25).
A second family, composed of S sequences, has a relatively well defined consensus: UACUANo lUGURCG...UYCACUAAGN, 2CCC (Table 16). Family 3 has a short, unreliable consensus motif (Table 16). In addition, there are approximately 12 orphans or ap~alel.tly unrelated sequences. Three of the orphan35 sequences were recovered at least twice (Table 16).

C. ~ffinitiP,S
The dissociation constants of representative ligands from Table 16 are shown in Table 17. These c~lrnl~tions assume two ligand binding sites per chimera.

CA 02223275 l997-l2-02 WO 96~1~70~ PCTAJS9f/vg~-5 S The affinity of random 2'-F RNA cannot be reliably det~rmin~cl but is estimated to be a~plo~ lately 10 ~M.
The dissociation constants range from 34 pM to 315 nM at 37 ~C. Binding affinity is not expected to be temperature sensitive since selection was at 37~C and 2'-F RNA forms thermal stable structures, but binding has not been tested at lower 10 L~ el~tules. For the most part, the extreme differences in affinity may be related to predicted secondary structure (Example 25).
The observed ~ffinities of the evolved 2'-F RNA ligands reaffirm our proposition that it is possible to isolate oligonucleotide ligands with ~ffinities that are several orders of m~gnihl~e 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 surfàce was tested by colllycLilion-flow cytometry eA~ Pntc with 20 human peli~he.~l blood lymphocytes. Lymphocytes were stained with 10 nM
FITC-conjugated DNA ligand FITC-LD201Tl (SEQ ID NO: 185) in the presence of increasing concentrations of unlabeled 2'-F ligands as d~sçrihe-l in Example 22,paragraph E. T .ig~n~15 LF1513 (SEQ ID NO: 321), LF1514 (SEQ ID NO: 297), LF1613 (SEQ ID NO: 331) and LF1618 (SEQ ID NO: 351) inhibited the binding 25 of FITC-LD201Tl in a concentration dependent manner, with complete inhibitionobserved at competitor concentrations of 10 to 300 nM. These results demonstratethat the 2'-F ligands are capable of binding cell surface L-selectin and suggest that the 2'-F ligands and LD201Tl bind the same or overlapping sites. The affinities of the fluoro lig~n~s, calculated from the coll,~Lition curves, range from 0.2 to 25 nM.
30 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 theDNA ligand LD201Tl (Kd = 3 nM). The reasonable agreement between the ~ffinities for punfied protein and lymphocyte L-selectin suggests that binding to lymphocytes is specific for L-selectin. These data validate the feasibility of using 35 irnmobilized, purified protein to isolate ligands against a cell surface protein.

Example 25 - Secondary Structure of High Affinitv 2'-F RNA Li~ands to L-Selectin In favorable inct~n~es, colll~ tive analysis of aligned sequences allows 40 deduction of secondary structure and structure-function relationships. If thenucleotides at two positions in a sequence covary according to Watcon-Crick base W O S5"tl~3 PCT/U',~/~54JS

5 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 ~ecl~lced secondary structure of family la ligands from co~ arative 10 analysis of 21 unique sequences is a hairpin motif (Figure 15) concisting of a 4 to 7 nucleotide tPrmin~l loop, a 6 base upper stem and a lower stem of 4 or more basepairs. The c- nCPncus terrninal loops are either a UWU tetraloop or a UUWVVU
pentaloop. Hexa- and heptaloops are relatively rare. The upper and lower stems are ~lelin~te~ by a 7 nucleotide bulge in the 5'-half of the stem. Four of the six base 15 pairs in the upper stem and all base pairs in the lower stem are ~ ol~ed 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 4or S base pairs long but can be ext~n-le(l While the sequence of the upper stem is strongly conserved, that of the lower stem is not, with the possible exception of the 20 YR' base pair adjacent to the intPm~l bulge. This base pair appears to covary with the 3' position of the 7 nucleotide bulge in a manner which lll;llilll;7eS the likelihood of e~ct~ntling the upper stem. Both the sequence (CUYARRY) and length (7 nt) of the bulge are highly conserved.
In terms of conlpalaLi~e analysis, the 7 nucleotide bulge, the upper stem and 25 the 5' and 3' positions of the tçrmin~l loop are most apt to be directly involved in L-selectin binding. Specifically, the 5' U and 3' U of the termin~l loop, the invariant GC and GA base pairs of the upper stem and the conserved C, U and A of the bulgeare the mostly likely c~n-litl~tes. The lower stem, because of its variability in length and sequence, is less likely to be directly involved. The importance of the bulge for 30 binding is ~uppol~ed by the poor affinity of ligand LF1512 (SEQ ID NO: 357; Kd =
315 nM); the simplest structure for this ligand is a UUW 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 single35 stranded bulge which does not have a highly conserved consensllc 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 40 to la and lb, a convincing consenslls secondary structure is not evident, perhaps due to incnfficient data. The most highly structured member of the family, LF1618 CA 02223275 l997-l2-02 WO 96'~5/0~ PCT~U~ ,1,5 S (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 lc forms a less 10 successful structure.
Predictions of minim~l 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 t~rmin~l loop, LF1514T1 (SEQ ID NO: 385) or these two elements plus the 7 nucleotide bulge sequence, LF1514T2 (SEQ ID
15 NO: 386), are not expected to bind with high affinity. On the other hand, there is a reasonable, but not rigorous, expectation that ligands tr~ln~ted at the base of the lower conc~nc.lc stem, LF1514T4 (SEQ ID NO: 387) and LF1807T4 (SEQ ID NO:
388), will bind with high affinity. In side by side col~lp~,sons, the ~ffinities of LF1514Tl and LF1514T2 for LS-Rg were reduced at least 100-fold in comp~ri~on 20 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 cc,l.cspol-~lPn~e bc~ween the predicted and observed truncate ~ffinities SU~pOlLS the postulated secondary structure.
Since the ssDNA ligand LD201T1 (SEQ ID NO: 185) and the adhesion 25 blocking anti-human L-selectin antibody DREG56 are known to bind to the lectin domain of L-selectin, colll~cLiLion between radio-labeled LF1807 (SEQ ID NO: 309) and either unlabeled DREG56 or unlabeled LD201Tl can serve to det~rmin~ if the 2'-F ligands also bind the lectin domain of purified LS-Rg. In these expprimen both DREG56 and LD201Tl gave concentration dependent inhibition of LF1807 30 binding. Complete inhibition was ~tt~in~rl with 300 nM Mab and 1 ~lM LD201Tl.The cor~petitols' affinities of LS-Rg, calculated from the co~ cLiLion curves, were in good agreement with their known ~ffinities 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 L-35 selectin m~ t~d adhesion. These results also reaff~m the proposition that theSELEX protocol, with S mM elution of bound oligonucleotides, ~crc~cllLially elutes ligands bound at or near the lectin domain's bound calcium.

CA 02223275 l997-l2-02 WO 96/1D70~ PCT/~3C'0~5 Example 26 ssDNA Ligands to Human P-Selectin PS-Rg is a chimeric protein in which the lectin, EGF, and the first two CRD
~om~ins of human P-selectin are joined to the Fc domain of a human G1 imm~lnoglobulin (R.M. Nelson et al., 1993, supra). Purified chimera is provided 10 by A.Varki. Soluble P-selectin is purchased from R&D Systems. Unless otherwise in~ljc~trcl, all materials used in the ssDNA SELEX against the P-selectin/IgG, chimera, PS-Rg, are i~lentir~l 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
15 antagonists to P-sçl~ctin are described in Examples 7 and 13.

Fxample 27 2'-F RNA Li~ands to Human P-Selectin The F.~pr~ t~l procedures outlined in this F.Y~mpl~ were used to identify 20 2'-F RNA ligands to human P-selectin as described in Examples 28-34.
F ~p~.; " ,~ l Procedures A) Materials PS-Rg is a rhim~ric protein in which the extracellular domain of human P-selectin is joined to the Fc domain of a hurnan G2 immllnoglobulin (Norgard et al., 25 1993, PNAS 90:1068-1072). ES-Rg and CD22~-Rg are analogous constructs of E-selectin and CD22~ 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 chi~ were provided by A.Varki. Soluble P-selectin was purchased from R&D Systems. Protein A
30 Sepharose 4 Fast Flow beads were purchased from Pharmacia Biotech. Anti-P-selectin monoclonal antibodies: Gl was obtained from Centocor. The 2'- 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 chemir~lc were purchased from commercial sources. Unless otherwise 35 in(lir~tecl experiments utilized HSMC buffer (1 mM CaC12~ 1 mM MgC12, 150 rnM NaCl, 20.0 mM HEPES, pH 7.4).

B) SELEX
The SELE~ procedure is described in detail in United States Patent 40 5,270,163 and elsewhere. The nucleotide sequence of the synthetic DNA t~mpl~te WO 96/40703 PCT/U' r - 09 ~5 5 for the PS-Rg SELEX was randomi7ed at 50 positions. This variable region was flanked by N8 5' and 3' fixed regions. The transcript 50N8 has the sequence 5' ggg~g~ g~ cgcucaa-SON-uucg~r~ga~gcur7~ ggC 3' (SEQ ID NO: 390).
All C and U have 2'-F ~ul~liLuLed for 2'-OH on the ribose. The primers for the PCR were the following:
- 10 N8 5' Primer 5'taatacg~tc~t~t~g~aga.~g~t~cgctcaa3' (SEQIDNO:
197) N8 3' Primer 5' gcctgttgtgagcctcctgtcgaa 3' (SEQ ID NO: 198) The fixed regions include primer ~nnP~ling sites for PCR and cDNA synthesis as well as a con~çnC~ls T7 promoter to allow in vitro transcription. The initial RNA
15 pool was made by first Klenow extP-rl-ling 1 nmol of synthetic single stranded DNA
and then transcribing the reslllting double str~n~l~Prl molPcllles with T7 RNA
polymerase. Klenow extension conditions: 3.5 nmols primer SN8, 1.4 nmols 40N8, lX 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 mPAi~ted synthesis of single stranded cDNA. These single-stranded DNA molecules were cc.l.v~lled into double-stranded transcription templates by PCR
amplification. PCR conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgC12, 1 mM of each dATP, dCTP, dGTP, and d l-l P, 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 8.0), 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-32P 2'-OH ATP.
The strategy for partitioning PS-Rg/RNA complexes from unbound RNA is essPnti~lly identical to the strategy ~Pt~ in Example 7 for ligands to L-selectin (Table 18).
In the initial SELEX rounds, which were performed at 37 ~C, the density of irnmobilized PS-Rg was 20 pmols/~ll of Protein A Sepharose 4 Fast Flow beads. Inlater rounds, the density of PS-Rg was reduced (Table 18), as nPede~1, 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 m~tt.ri~l from only one PS-Rg density was carried forward.
Before each round, RNA was batch adsorbed to 100 ~Ll of protein A
sepharose beads for 1 hour in a 2 ml siliconized column. Unbound RNA and RNA
40 eluted with minim~l washing (two volumes) were combined and used for SELEX

W O ~ 0703 PCT~USg~v~155 S input m~t~ri~1 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 (500 111 HSMC/wash). Bound RNA was eluted as two fractions; first, bound RNA was eluted by incub~ting and washing columns with S mM EDTA in HSMC
10 without divalent cations; second, the r~ lg elutable RNA was removed by incubating andlor washing with 50 mM EDTA in HSMC without divalents. The pelcelltage 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 idçnti~lly to the SELEX beads to ~lete- ...i..~ background binding. All unadsorbed, 15 wash and eluted fr~tiQnc were counted in a Rec~m~n LS6500 sçintill~tion counter in order to lllonilor each round of SELEX.
The eluted fractions were ~r~cesse-l for use in the following round (Table 18). After P~GC jP;~ ;llg with 300 mM Sodium Acetate pH 7 in ethanol (2.5 volumes), the RNA was lesu~Gllded in 80 ~l of H20 and 40 ~Ll were reverse 20 tr~nc~r~ into cDNA by AMV reverse transcriptase at 48 ~ C for 30 minntes, in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6 mM Mg(OAc)2, 10 rnM DTT, 200 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unitl~Ll AMV RT. Transcripts of the PCR product were used to initiate the next round of SELEX.

25 C) Nitrocellulose Filter Binding Assay As described in SELEX Patent Applications, a nitrocellulose filter partitioning method was used to ~let~-~ ., .i . .e the affinity of RNA ligands for PS-Rg and for other proteins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 ,um pore size, Millipore) were pl~ed on a vacuum manifold and washed with 230 ml of HSMC buffer under vacuum. Reaction mixtures, CO~ g 32p labeled RNA pools and unlabeled PS-Rg, were incubated in HSMC for 10 - 20 min at 4 ~C, room t~ Ul~ or 37 ~C, filtered, and then imm~ tPIy washed with 4 rnl HSMC
at the same telll~l~ e. The filters were air-dried and counted in a Beckm~n LS6500 liquid scintill~tion counter without fluor.
PS-Rg is a dimeric protein that is the e~eSSiOn product of a recombinant gene constructed by fusing the DNA sequence that encodes the extr~çll~ r domainsof human P-selectin to the DNA that encodes a human IgG1 Fc region. For affinityc~lrul~tions, one ligand binding site per PS-Rg monomer (two per dimer) were ~csllm~-~l The monomer concentration is defined as 2 times the PS-Rg dimer WO 96.'1~i~3 PCT/U~, !09~'S

5 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 10 either a BamHI or a HinDm restriction ~n~onllclease 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, Gaithc~a7~ g~ MD). Plasmid DNA was prepared according to the AlkAline hydrolysis method (PERFECTprep, 5'-3', Boulder, CO).
15 Approximately 50 clones were seq-lPnce-l using the Sequenase protocol (Amersham, Arlington ~leip,htc, IL). The resulting ligand sequences are shown in Table 19.

E) Boundary E~ lLa The minimAl high affinity sequence of individual ligands was d~te~ .";i~rl by 20 boundary e~e.illle~ts (Tuerk et. al. 1990, J. Mol. Biol. 213: 749). Individual RNA lig~ntlc, 32P-labeled at the 5'-end for t'ne 3' boundary and 32P-labeled at the 3'-end for the 5' boundary, are hydrolyzed in 50 mM Na2CO3 pH 9 for 8 minutes at 95~C. The resulting partial hydrolysate contains a population of end-labeled molecules v~hose hydrolyzed ends correspond to each of t'ne purine positions in the 25 full length molecule. The hydrolysate is incubated with PS-Rg (at concentrations 5-fold above, below and at the measured Kd for the ligand). The RNA concentration is ci~nific~nrly lower than the Kd. The reaction is incub~tecl at room tt;lll~clature for 30 minlltes, filtered, and then imm~ t~ ly washed with 5 ml HSMC at the same telll~l~ture. The bound RNA is extracted from the filter and then electrophoresed 30 on an 8% den~tllring 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 tne 2'-F pyrimidine RNA ligands to 35 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 synth~oci7e(1 each with three mixed positions. A mixed position is defined as a 2'-OH purine nucleotide within the RNA which has been 40 synthesized with 2: l ratio of 2'-OH:2'-OMe. Since the coupling efficiency of 2'-WO 9f ' ~0703 PCI/US9C~'~S ~55 5 OH phosphor~mi-lites is lower than that of 2'-OMes, the reslllting RNA has 25-50 % 2'-OH at each mixed position. 32p 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 telllp~ldLul~ for 30 minllt~s, filtered, and then imm~ t~ly washed with S ml HSMC at the same telllpc;ldlul~. The bound RNA
10 (Selected RNA) is extracted from the filter and then hydrolyzed with 50 mM
Na2C03 pH 9 for 8 minllt~s, at 95~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% rl~n~tllrin~ gel adjacent to Unselected RNA.
To ~L~ the affect on binding affinity of 2'-OMe s~lb~LiLuLion at a 15 particular position, the ratio of int~ncitiçs of the Unselecte(l 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 e~l,r,;, . ,~ntc in which the position is not mixed. If the Unselected:Selected ratio of the mixed position is si~nifi~ntly greater than that when the position is not mixed, 20 2'-OMe may increase affinity. Conversely, if the ratio is ~ignifi~ntly less, 2'-OMe may decrease affinity. If the ratios are not significantly dirr~,.el~t, 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 expl-rim~nt~ with humanplatelet suspensions. Whole blood from normal volunteers was collected in Vacutainer 6457 tubes. Within 5 minutes of collection, 485 !ll of blood was stim~ ted with 15 ~11 Bio/Data THROMB~-EX for 5 Ill;llllltos at room telll~erd~
A 100 ~Ll aliquot of ~timnl~t~l blood was transferred to 1 rnl of BB- (140 rnM NaCl, 20 mM HEPES pH 7.35, 5 mM KCl, 0.01% NaN3) at 4~C and spun at 735 x g for 5 minllt~os This step was repeated and the resulting pellet was re-suspended in 1 ml of BB+ (140 mM NaCl, 20 rnM HEPES pH 7.35, 5 mM KCl, 0.01% NaN3, 1 mM
CaC12, 1 mM MgC12) at 4~C.
To detect antigen expression, 15 !11 BB+ containing FITC conjugated anti-CD61 or PE conjugated anti-CD62 antibody (Becton Dickinson) was incubated for 20-30 minl~tes at 4~C with 10,ul of platelet suspension. This was diluted to 200 ~1 with 4~C BB+ and analyzed on a Becton Dickinson FACSCaliber using 488 nm WO 95' .C703 PCTAJ~ ûr 'S~

5 excitation and FLl (530 nm emission) or FL2 (580 nm emission) with the m~l-hin.~.
live gated on platelets. Between 1000 and 5000 events in this gate were recorded.
To detect oligonucleotide ligand binding, 15 ~Ll BB+ cont~inin~ ligand conjugated to either FITC or biotin was incllb~ted 20-30 minut~c at 4~C with 10 Jll platelet sllcp~nsion. The FITC-ligand incubations were diluted to 200 ,ul with BB+
10 and analyzed on a FACSCaliber flow cytometer. The biotinylated-ligand reactions were incubated with streptavidin-phycoerythrin (SA-PE) (Becton Dickinson) for 20minllt~s at 4~C, before dilution and analysis. Wash steps with 500 ~11 BB+ and 700 x g spins have been used without colllplvlllising the quality of the results.
The specificity of binding to P-selectin (CD62P) expressed on platelets was 15 tested by competition with the P-selectin specific blocking monoclonal antibody, Gl. 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 20 PS-Rg to sialyl-LewisX was tested in competitive ELISA assays (C. Foxall et al., 1992, supra). For these assays, the wells of Corning (25801) 96 well rnicrotiterplates were coated with 100 ng of a sialyl-LewisXlBSA conjugate, air dried overnight, washed with 300 ~11 of PBS(-) and then blocked with 1% BSA in HSMC
for 60 min at room tclll~erdture. RNA ligands were incubated with PS-Rg in 25 HSMC11% BSA at room temperature for 15 min. After removal of the blocking solution, 50 ~11 of PS-Rg (lOnM) or a PS-Rg (lOnM)/RNA ligand rnix 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 )11 of PBS(-) and then probed with HRP conjugated anti-human IgG, at room tc..~el~ture to quantitate PS-Rg binding. After a 30 minute incubation at room t~.pe.dLu~c in the dark with OPD
peroxidase substrate (Sigma P9187), the extent of PS-Rg binding and percent inhibition was det~rmin~d from the OD4so.

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 40 estim~t~d to be approximately 2.5 ,uM. An eight-fold difference was observed in the CA 0222327~ 1997-12-02 WO ~6/lD703 PCT/U' 3~5 ~SS

5 RNA elution profiles with 5 rnM EDTA from SELEX and background beads for rounds 1 and 2, while the 50 rnM 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 5mM eluate was processed for the following round. To increase the stringency of 10 selection, the density of immobilized PS-Rg was reduced five fold in round 2 and again in round three without greatly redllcin~, the fraction eluted from the colurnn.
The density of imrnobilized PS-Rg was further reduced 1.6-fold in round 4 and rem~in~d 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 pools15 gradually evolved biphasic binding char~ctPricti~s.
Binding e~ with 12th round RNA revealed that the affinity of the evolving pool for P-selectin was not te~ eldLule sensitive. BuLI~ seqllPn~ing of 2nd, 6th, 11th and 12th RNA pools revealed noticeable non-randomness by round twelve. The 6th round RNA bound monophasically at 37 ~C with a dissociation 20 COllSL~.t of approximately 85 nM, while the 11th and 12th round RNAs bound biph~cic~lly 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~ /Mg~., plus 2 mM EDTA). The 12th round pool showed high specificity for 25 PS-Rg with measured Kd's of 1.2 ,uM and 4.9 ,uM for ES-Rg and LS-Rg, respectively.

B. RNA Sequences In Table 19, ligand sequences are shown in standard single letter code 30 (Cornish-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.
35 Sequences that were isolated more than once, are indicated by the parenthetical number, (n), following the ligand isolate number. These clones fall into five sequence families (1-5) 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 40 consensus sequence is composed of two variably spaced sequences, WO 96/40703 PCT/U~ 0~ ~'5 5 CUCAACGAMC and CGCGAG (Table 19). In 11 of 13 ligands the CUCAA of the consensus is from 5' fixed sequence which consequently l~lillill~;7~:,C variability and in turn reduces confidence in interpreting the i~ olL~lce of CUCAA or the paired GAG (see Example 27).
Farnilies 2-5 are each represented by mllltiple isolates of a single sequence 10 which precludes detçrmin~tion of conce~cllc sequences.

D. ~ffinities The dissociation constants for representative lig~n~l$, including all orphans, were ~t~t~ l;n~cl by nitrocellulose filter binding e~ ntc and are listed in Table 15 20. These calculations assume two binding sites per chimtor~ The affinity of random RNA is estim~t~ to be approximately 2.5 ~M.
In general, ligands bind monophasically with dissociation ct)nct~ntc ranging from 15 pM to 450 pM at 37 ~C. Some of the highest affinity ligands bind biph~ci~lly. Full length ligands of f~milie,c 1-4 show no t~l~e~dLule dependence.
20 The observed affinities subst~nti~te the proposition that it is possible to isolate oligonucleotide ligands with ~ffiniti~,c that are several orders of m~gnitlltle greater than that of carbohydrate lig~nrlc Example 29 Specificity of 2'-F RNA Ligands The affinity of P-selectin ligands to ES-Rg, LS-Rg and CD22~-Rg were clet~rmin~ by nitrocellulose partitioning. As inllic~t~o~l 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 104-fold lower than for PS-Rg. Binding above background is not observedfor CD22,13-Rg at the highest protein concentration tested (660 nM), in-lic~ting 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 confilms the proposition that SELEX ligands will have greater specificity than carbohydrate ligands.

Example 30 Inhibition of Binding to sialyl-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 9~'5C703 PCT/US9~/~9~1S5 consequently, are expected to compete with sialyl-LewisX for binding. In colllp~ilion assays, the selected oligonucleotide ligands competitively inhibit PS-Rg binding to immobilized sialyl-LewisX with IC50s ranging from 1 to 4 nM (Table 20). Specifically, ligand PF377 (SEQ ID NO: 206) has an IC50 of approximately 2 nM. Complete inhibition is ~tt~inP~1 at 10 nM ligand. This result is typical of high affLnity ligands and is reasonable under the experimt-nt~l conditions. The IC50s of ligands whose Kds are much lower than the PS-Rg concentration (10 nM) are ~imited by the protein concentration and are expected to be a~ o~ lalely one half the PS-Rg concentration. The specifi~ity of competition is demonstrated by the inability of round 2 RNA (Kd~ 1 ,uM) to inhibit PS-Rg binding to immobili~ed sialyl-LewisX. These data verify that 2'-F RNA ligands are functional antagonists of PS-Rg.

Example 3 1 Secondary Structure of High Affinitv Ligands In favorable in~t~nres, colll~a~dtive analysis of aligned sequences allows deduction of secondary structure and structure-function relationsnips. If the nucleotides at two positions in a sequence covary according to Watson-Crick basepairing 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 bedirectly involved.
Comparative analysis of the family 1 ~lignm~nt suggests a hairpin motif, the stem of which contains three asymmetrical intemal 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 sarne 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 telll~inus of the stem.
Conclusions regarding a direct role of the terminus in binding are lelllpeled 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 p~"", ll~tion 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/U'3C.~ 55 Example 32 Boundary Experiments Boundary e~ r~ 'I)tc 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 10 composite boundary species vary in size from 38-90 nucleotides, but are 4045 nucleotides in family 1. ~ffinities of these truncated ligands are shown in Table 22.
In general, the truncates lose no more than 10-fold in affinity in colllp~ison to the full length, effectively inhibit the binding of PS-Rg to sialyl-Lewisx and m~int~in binding specificity for PS-Rg (Table 22). These data validate the boundary method 15 for identifying the minim~l high affinity binding elerr Pnt of the RNA lig~n-lc Example 33 Binding of 2'-F RNA Ligands to Human Platelets Since the P-selectin ligands were isolated against purified protein, their 20 ability to bind P-selectin presented in the context of a cell surface was d~tçrrnined in flow ~;ylo~ y eXp~rim~ntc~ with activated human platelets. Platelets were gated by side scatter and CD61 expression. CD61 is a col~sLiLuLively expressed antigen on the surface of both resting and activated platelets. The e~lt;ssion of P-selectin was monitored with anti-CD62P monoclonal antibody (Becton Dickinson). The mean 25 fluorescence intensity of activated platelets, stained with biotintylated-PF377s 1 (SEQ ID NO: 223)/SA-PE (Example 27, paragraph G), is 5 times greater than that of similarly stained resting platelets. In titration ~ ntc, half maximal fluorescence occurs at approximately 50 pM PF377s 1 (EC50) which is consistent with its equilibrium dissociation constant, 60 pM, for PS-Rg. Binding to platelets is 30 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 pl~t~letc is divalent cation dependent; and most importantly 3) that 35 binding is inhibited by the anti-P-selectin adhesion blocking monoclonal antibody Gl, 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 91~/~C703 PCT/U' ,G/0~ ~5 Example 34 2'-O-Methyl Substitution Experiments 2'-OMe purine ~ubsLiLuLions were performed on ligand PF377sl (SEQ ID
NO:223) as described in F.~mple 27 paragraph F and the results are shown in Table 23 . The data in-lirat~o t_at 2'-OMe purines at positions 7-9, 15, 27, 28 and 31 10 çnh~nre binding while substitutions at positions 13, 14, 16, 18, 21 22, 24, and 30 have little or no affect on affinity. Thus it appears that up to 15 positions may be ç(l with only slight losses in affinity. In partial confirm~tion of this e~ecta~ion, the affinity of 377sl.cim-l1t~neously ~ubsLiLuLcd with 2'-OMe purines at 11 positions (~ I6, SEQ ID N 0: 235) is 250 pM (Table 22).
Example 35 2'-NH~ RNA T .ig~nflc to Human P-Selectin The Ç~l r~ ;IIlr.ll;ll procedures described in this Example are used in Examples36-38 to isolate and chala~ 7e 2'-NH2 RNA ligands to human P-selectin 20 F~ r~ l ~ocedulcs A) Materials Unless otherwise inflir,~te~l all m~tPri~l~ used in the 2'-NH2 RNA SELEX
against the P-selectin/IgG, c~ à, PS-Rg, were iclentir~l to those of FY~rnrle 27.
The 2'-NH2 modified CTP and UTP were prepared according to Pieken et. al.
25 (1991, Science 253:314-317). The buffer for SELEX e~e. ;Illr.l~lc was 1 m~
CaCl2, 1 mM MgC12, 150 mM NaCI, 10.0 mM HEPES, pH 7.4.

B) SELEX
The SELEX procedure is described in detail in US patent 5,270,163 and 30 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 5' and 3' fixed regions. The transcript 50N8 has the sequence 5' gggag~r~ga~ r gcucaa-50N-uucgacaggaggc~c~ c~g~c 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 35 following:
N8 5' Primer 5' taatacg~rtc~ct~t~ggg~g~r~g~t~rgctcaa 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 identic~l -40 to those described 2'-F ligands in Example 27, except that transcription reactions WO 96J4~703 PCT/US9~5 ~'5 5 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 10 C, a nitrocellulose filter partitioning method was used to cl~te~ 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 (Fx~mple 7C) was used for these e~ c Binding data were analyzed as described in Example 7, paragraph C.

D) Cloning and Seq..en~ing Twelfth round PCR products were re-amplifit d with primers which contain either a BamHI or a HinDm restriction endon~ ce recognition site.
Applo~ ly 75 ligands were cloned and sequenced using the procedDs 20 described in Example 7, paragraph D. The rçslllting sequences are shown in Table 25.

E) Cell Binding Studies The ability of evolved ligand pools to bind to P-selectin presented in the 25 context of a cell surface was tested in flow cytometry ~,~ . ;",~"l~ with human platelet ~u~,~nsions as described in Example 7, paragraph E.

Example 36 2'-NH~ RNA T.igantls to Human P-Selectin 30 A. SELEX
The starting 2'-NH2 RNA pool for SELEX, randomized 50N8 (SEQ ID
NO: 248), cont~inerl a~plv~i,l,ately 1015 molecules (1 nmol 2'-NH2 RNA). The ~licsoci~tion constant of randomized RNA to PS-Rg is estim~tç~l to be approximately 6.4 ~lM. The SELEX protocol is outlined in Table 24.
The initial round of SELEX was perforrned at 37 ~C with an PS-Rg density of 20 pmolJ~Ll of protein A sepharose beads. Subsequent rounds were all at 37~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.
40 This scheme was continued through round 6. Starting with round seven only the 5 CA 0222327~ 1997-12-02 WO 96/40703 PCT/U~ 15 S rnM eluate was processed for the next round. To increase the stringen~ y of selection, the density of irnmobilized 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 G~ with 12th round RNA revealed that the affinity of the 10 evolving pool for P-selectin was te~ aLulG sensitive despite performing the selection at 37~C, (Kds: 13 pM, 91 pM and 390 pM at 4 ~C, room tGlll~Gl~ G and 37 ~C, lGs~ecli\rely~. BuLk seq~ nring of RNA pools in~ tecl dramatic non-randomness at round 10 with not many visible changes in round 12. T .igantlc were cloned and sequenced from round 12.
15 B. 2'-NH2 RNA Sequences In Table 25, the 2'-NH2 RNA ligand sequences are shown in standard single letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030)(SEQ nD NOS:
251-290). The evolved random region is shown in upper case letters in Table 25.
Any portion of the fixed region is shown in lower case letters. By definition, each 20 clone in~ es both the evolved sequence and the associated fixed region, unless speçifir~lly stated otherwise. From the twelfth round, 40/61 sequenced ligands were unique. A unique seqll~n~ e is operationally defined as one that differs from all others by three or more nucleotides. Sequences that were isolated more than onceare inr~ t~ by the parenth~tir l number, (n), following the ligand isolate ~ lbel.
25 T ig~ntl~ from farnily 1 domin~te the final pool cc-nl~;";,-g 16/61 sequences, which are derived from multiple line~gses. Families 2 and 3 are represented by slight mutational variations of a single sequence. Sequences labeled as "others" do nothave any obvious ~imil~rities. Family 1 is characterized by the concçncuc sequence GGGAAGAAGAC (SEQ nD NO: 291).

C. ~ffiniti~s The dissociation constants of representative ligands are shown in Table 26.
These calculations assume two RNA ligand binding sites per ch"..e.a. The affinity of random 2'-NH2 RNA is e~tim~te~l to be approximately 10 ~
At 37~C, the dissociation constants range from 60 pM to 50 nM which is at least a lx103 to lx105 fold ~lllplov~...ent over randomized 2'-NH2 RNA (Table 26). There is a marked t~.pe.~lu.e sensitivity for Clone PA350 (SEQ ID NO:
252) with an increase in affinity of 6 fold at 4~C (Table 26). The observed affinities of the evolved 2'-NH2 ligand pools reaffirrn our proposition that it is possible to wo g~'~O703 PCT/U' 3GJ~S1 5 isolate oligonucleotide ligands with ~ffinitit~.s that are several orders of m~gni greater than that of carbohydrate lig~ntlc.

Example 37 Specificit,v of 2'-NH~ RNA Ligands to P-Selectin ~ 10 The affinity of clone PA350 (SEQ ID NO: 252) for LS-Rg and ES-Rg was clete. . ~ .i . .~d 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 SxlO5-fold less than for PS-Rg. Binding above background is not observed for CD22~-Rg intli~ting that ligands neither bind the 15 Fc domain of the çhimtoric constructs nor have affinity for unrelated sialic acid binding sites.
The specifi~ity of oligonucleotide ligand binding contrasts sharply with the binding of cognate carbohydrates by the selectinc and reconfilms the proposition that SELEX ligands will have greater specificity than carbohydrate li~ntlc 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 25 flow ~ylOllletly eAy~ ntC as described in Example 23, paragraph G.
The specificity of FITC-PA350 for binding to P-selectin was tested by coll.yelilion el~ye. ;",.~1 .t.~i in which FITC-PA350 and unlabeled blocking monoclonal antibody G1 were cimlllt~nPously added to stiml-l~ted platelets. G1 effectively colll~tes with ~l l C-PA350 for binding to platelets, while an isotype m~tchPd 30 control has little or no effect which demonstrates that ~l l C-PA350 specific~lly 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 ~l l C-PA350 is dependent on divalent cations; at 10 nM ~l l C-PA350 activated platelets are not stained in 35 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.

wo 9~ 703 PCT/US~ ~9155 Example 39 Inhibition of P-selectin Binding to Sialyl LewisX
In competition assays, ligands PA341 (SEQ ID NO: 251) and PA350 (SEQ
ID NO: 252) co~ Litively inhibit PS-Rg binding to immobilized sialyl-LewisX withICSOs ranging from 2 to 5 nM (Table 26). This result is typical of high affinity10 ligands and is reasonable under the e~ nt~l conditions. The ICSOs of ligands whose Kds are much lower than the PS-Rg concentration (10 nM) are limited by theprotein concentration and are expected to be a~,o~illlately one half the PS-Rg concentration. The specificity of competition is demonstrated by the inability of round 2 RNA (Kd~ 1,uM) to inhibit PS-Rg binding to immobilized sialyl-LewisX.
15 These data verify that 2-NH2 RNA ligands are functional antagonists of P-selectin.

Example 40 2'-NH~ RNA Li~ands to Human E-Selectin ES-Rg is a chim~ric protein in which tne extracellular domain of human E-20 selectin is joined to the Fc domain of a human G1 immllnoglobulin (R.M. Nelson et al., 1993, supra). Purified chimera were provided by A.Varki. Unless otherwise nflic~t~ all m~t~ri~lc used in this SELEX are similar to those of Examples 7 and13.
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.

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CA 0222327~ 1997-12-02 WO 9G/~703 PCT/U' 3C1~3 1-5 Table 3 Dissociation Constants of RNA Ligands to WGA

Liga~dSEQ IDNO: Kd Family 1 11.8 4 9.2nM
11.4 5 32nM
11.35 7 90nM
11.5 8 44nM
11.26 9 38nM
11.19 10 22nM
11.15 11 54nM
11.34 12 92nM
6.8 13 11nM
6.9 14 396nM
6.23 ~ 15 824nM
6.14 17 <5%
Family 2 11.12 18 15.2nM
11.24 19 19.4nM
11.27 20 30nM
~1.32 21 274nM
11.6 22 702nM
Family 3 11.13 23 c5%
11.23 24 ~5%
6.3 25 120nM
6.2 27 ~5%
6.34 28 ~5%
6.35 29 ~5%
6.5 30 678nM
6.16 31 ~5%
6.19 32 74nM
Family 4 11.2 36 62nM
11.33 37 ~5%
11.28 38 9.2nM
11.7 39 16nM

W O g~703 PCT/U~r.!0~'155 TABLE 3 (Page 2) Ligan~SEQ ID NO: Kd Family 5 11.2 40 1.4 nM
Family 7 6.27 44 56 nM
11.3 45 410nM
Family 8 6.6 47 ~5%
Family 9 6. 11 48 <5%
Orphans 11.3 54 56 nM
11.29 55 32 nM

The Kds of ligands that show < 5 ~ binding a. 1 ~M WGA
is esti~ated to be > 20 ~M.

WO 9~"~703 PCT/US9~ 9 ~rS

Table 4 Specificity of RNA Ligands to WGA

Kds for N-acetyl-glucosamine Binding Lectins LECTIN Ligand 6.8Ligand 11.20Ligand 11.24 (SEQ ID NO:13) (SEQ ID NO:40) (SEQ ID NO:19) Triticum vulgare (WGA) 11.4 nM 1.4 nM 19.2 nM
Canavalia ensiforrnis (Con A)** <5%* <5%* <5%*
Datura stramonium <5%* 11.2 ~M <5%*
Ulexeuropaeus (UEA-II) 4.4 IlM 2.2 ~lM ~5%*

* Less than 5% binding at 1 IlM protein; estimated Kd > 20 ~LM
** succinylated Con A

T~RT,F. 5 I NH I ~TTION OF RNA LIOEaND ~lNL~l~
TO W~AT GERM AGGUL~

Liaand SEO ID NO: ComDetitor ICso (!lM) M~ In}lib Rc (~M) 6 . 8 13 (GlcNAc) 3 95 > 95 %10 . 9 11.20 40 (GlcNAc) 3 120 ~ 95 %8.4 11. 24 19 (GlcNAc) 3 120 > 95 %19 . 4 Rc is the dissociation constant of ~GlcNAc)3 calculated ~rom these data, assuming competitive inhibition and two RNA ligand binding sites per dimer.

CA 02223275 l997-l2-02 W O ~ C7~3 PCT/U~,~'0 Table 6 I NH I~TTION OF ~ ~ D ~ TED A~r-TullrL~TION
OF .~ H~ ~ V ~y 'l~LKCK~r~ES

Tnh;h; tory ~on~sn~ation (~M) Tnh; h; tor SEO ID NO: C~u~lete Partial 6.8 13 0.5 0.12 .20 40 0.5 0.12 11 . 24 19 * 2 (GlcNAc) 3 8 2 GlcNAc 780 200 * Complete inhibition of agglutination by ligand 11. 24 was not observed in this experiment.

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WOg~"C703 PCT/U~ '0~55 TARRT~ g Dis~oc;~tion Constants of 2' N~ RN~ Ligands to L-Selectin*

Li~andSEO ID NO: 4 ~C km TemD
F~;ly I
F13.32 67 15.7 nM 14.9 nM
F13.48 74 15.9 nM 9.2 nM
F14.9 71 8.2 nM 15.4 nM
F14.21 72 2.3 nM 15.9 nM
F14.25 73 1300 nM
Family II
F14.12 78 5.8 pM 1.7 nM
(0.68) (0.62) 16.2 nM 94 nM
F14.20 77 58 pM 1.0 nM
(0.68) (0.28) 60 nM 48 nM
F~;ly III
F13.57 89 3.0 nM 75 nM
F~ly V
F13.55 93 62 pM 1.5 nM
Family VI
F14.53 95 97 pM 142 nM
(0.65) 14.5 nM
F14.27 94 145 nM
F~ily VII
F13.42 96 2.0 nM 5.5 nM
F13.51 97 8.8 nM 18 nM

WO 9~'1C~03 PCT/US96/09455 ~RT.~ 9 ( Page 2 ) Liaa~dSEO ID NO:4 ~C Rm Te~

F;~m; ly X
F14.2 101 1.8 nM 7.2 nM
F14.13 102 1.3 nM
(0.74) 270 nM
Orphans F13.59 116 < 5% < 5%
F14.70 117 2.0 nM 7.8 nM
(0.75) (0.58) 254 nM 265 nM
* Kds of monophasic binding ligands are indicated by a single number; the high affinity Kd (ie., Kdl), the mole fraction binding with Kdl~ and the low affinity Kd (ie-, Kd2) are presented for biphasic bi n~; ng ligands.

W096/40703 PCT/U',~'~Sl~S

~RT.~ 10 Specificity of 2r NE~ RN~ Ligands to L-Selectin*
Liaand SEO ID NO: LS-Ra ES-R~ PS-Ra CD22-Ra Fam;ly I
F13.32 6715.7 nM < 5% 17 ~M < 5%
F13.48 7415.9 nM < 5% 720 nM < 5%
F14.9 718.2 nM < 5% < 5%
F14.21 722.3 nM 2.6 ~M < 5%
F14.25 731300 nM
Fam;ly II
F14.12 78 60 pM 47 nM 910 nM < 5%
F14.20 77 58 pM 70 nM < 5%
(0.68) 60 nM
Fa~;ly III
F13.57 89 3.0 nM 2.7 ~M < 5%
Fa~; ly V
F13.55 93 62 pM 49 nM 5.8 ~M < 5%
Fa"l; ly VI
F14.53 95 97 pM 355 nM 5.2 ~M < 5%
(0.65) 14.5 nM
Fam;ly VII
F13.42 96 2.0 nM 4.4 ~M < 5%
F13.51 97 8.8 nM 2.0 ~M
F2mily X
F14.2 101 1.8 ~M 1.9 ~M 450 nM < 5%

wo s~ a70~ PCT/U~

Table 10 ( Page 2 ) Liaand SEO ID NO: LS-Ra ES-Ra PS-Ra CD22-Ra OrphaIls F13.59 116 < 5% < 5% < 5%
F14.70 117 2.0 nM 5.9 ~M < 5%
(0.75) 254 nM

* Dissociation constants were determined at 4~C in HSMC buffer.
When < 5% binding was observed at the highest protein concentration, the Kd is estimated to be > 20 ~M.

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~,a a aa ~a a aaaaaaaaa CA 0222327~ 1997-12-02 W 0 96/40703 PCTrUSg'/05'-5 Table 13 Dissociation Constants of ssDNA Ligands to L-Selectin Room Li~and SEO ID NO: Temperature 37 ~ C
Family 1 LDlll 149 330 pM 11.8 nM
LD128 148 310 pM 1.8 nM
LD108 155 160 pM 8.5 nM
LD112 135 300 pM 23.2 nM
LD137 136 520 pM 0.65 nM
LD139 150 210 pM 6.8 nM
LD145 130 920 pM 8.8 nM
LD179 137 180 pM 590 pM
LD182 138 130 pM 2.0 nM
LD183 131 170 pM 1.O nM
LD193 140 88 pM 970 pM
LD201 142 110 pM 1.2 nM
LD204 129 100 pM 3.7 nM
~D208 155 110 pM 380 pM
LD227 134 43 pM 160 pM
LD230 132 57 pM 260 pM
LD233 146 110 nM 380 pM

Family 2 LD181 157 84 pM 1.8 nM

Family 4 LD122 159 1.8 nM 2.1 nM
LD174 158 43 pM 370 pM
LD239 160 170 pM 1.6 nM

Family 5 LD109 161 190 pM 9.6 nM
LD127 162 1.0 nM 890 pM

W09"~70~ PCT/U'r~09'~

TABLE 13 ( Page 2 ) Room LiaandSEO ID NO: TemPerature 37 ~ C

Family 6 LD196 163 130 pM 3.4 nM

Family 7 LD206 165 330 pM 6.0 nM

Orphans LD102 167not determined 7.9 nM
LD214 166 660 pM 8.4 nM

Round 15 Pool 160 pM 660 pM

LD201T1* 4.8 nM
LD201T3* 43 nM

* LD201T1 and LD201T3 were made by solid state synthesis; the Kd of the synthetic full length LD201 control was 3.8 nM while that of enzymatically synthesized LD201 was 1.8 nM.

WO 96/40703 PCT/U', ' ~9 t~5 Table 14 Specificities of ssDNA Ligands to L-Selectin*
Li~andSEQ TD NO: LS-R~ ES-R~ PS-R~
Family 1 LD111 149 1.1 nM 1.2,uM 840 nM
LD201 142 110 nM 37 nM 1.0 ~lM
LD204 129 450 pM 1.5 ,uM 2.9 IlM
LD227 134 64 pM 33 nM 560 nM
LD230 132 44 pM 19 nM 600 nM
LD233 146 120 pM 39 nM 420 nM
Family 2 LD181 157 200 pM 37 nM 1.6 ~M
Family 4 LD122 159 340 pM 400 nM 420 nM
LD174 158 46 pM 28 nM 380 nM
Family 5 LD127 162 250 pM 1.3,uM 780 nM
Family 6 LD196 163 220 pM 50 nM 3.4 IlM
Family 7 LD206 165 120 pM 100 nM 600 nM

*Kds were determined at room temperature. In assays with 700 nM CD22 B-Rg and 1.4 ~LM WGA less than l~o and 3% binding, respectively, was observed for allligands suggesting that the dissociation constants are greater than 100 ~LM for these proteins.

CA 0222327S l997-l2-02 W O 9"~703 PCT/U~ 4~5 Table 15 Summary of Selection Conditions and Results from 2'F RNA Human L-selectin SELEXes 30n7 2'Fluro SELEX
-SELEX Total Total Temp. % Bound % 5mMEDTA Kd(nM) Round RNA Protein Time, LS-R~ EDTA Si~nal/
pmoles pmoles Vol. Sites Eluted Bk~nd 630 100 37~C. 15' 101l1 0.7 0.1 20 2 656 100 37~C. 15' 10111 2.8 0.4 24 3 608 100 37~C. 15' 10~11 11.6 1.9 68 10000 4 193 20 37~C. 15' 10!11 7.4 0.8 24 193 20 37~C. 15' 10111 19.7 2.1 17 850 6 86 10 37~C. 15' lO,ul 15.7 1.9 8 360 7 17 2 37~C. 15' 10111 12.1 1.4 3 8 17 2 37~C. 15' 10!11 55.1 6.6 2 9 19 2 37~C. 15' 1O~LI 40.1 4.2 4 18 2 37~C. 15' 101l1 28.4 3.3 3 3 11 103 12.5 37~C. 15' 50~11 647.7 8.3 65 11 27 2.5 37~C. 15' 50~L1 63.1 5.9 3 0.5 12 89 5 37~C. 15' 50~L1 53.2 3.0 7 12 79 5 37~C. 15' 50111 54.8 3.5 65 0.4 40n7 2'Fluro SELEX
SELEX TotalTotal Temp, % Bound % 5mM SiS~nal/ Kd(nM) Round RNA Protein Time! LS-R~ EDTA Bk~nd pmoles pmoles VoR Sites Eluted EDTA
677 100 37~C. 15' 1O~LI 1.8 0.3 31 2 659 100 37~C. 15' 10~11 5.8 0.9 19 3 499 100 37~C. 15' 10~11 9.6 1.9 25 10000 4 187 20 37~C. 15' 101l1 4.3 0.5 7 179 20 37~C. 15' 10~11 19.7 2.2 8 1024 6 89 10 37~C. 15' 10111 17.7 2.0 12 240 7 19 2 37~C. 15' 10,ul 17.3 1.8 2 8 17 2 37~C. 15' 10111 78.9 10.4 5 9 19 2 37~C. 15' 10~11 36.5 4.1 3 18 2 37~C. 15' 10~11 14.1 2.3 2 0.9 11 99 12.5 37~C. 15' 50~1 60.3 7.7 16 11 22 2.5 37~C. 15' 50~L1 90.1 10.4 18 0.3 12 89 5 37~C 15' 50111 53.2 3.0 7 12 92 5 37~C. 15' 50~11 92.2 5.0 80 0.1 CA 0222327~ 1997-12-02 WO 9~"~703 PCT/U~5~ S~5 Table 15 (Page 2) 30n7 Primer Competition Counter-SELEX
SELEX Total TotalTemp. % Bound % 5mMEDTA Kd(nM) Round RNA Protein Time. LS-R~ EDTA Si~nal/
pmoles pmoles Vol. SitesEluted Bk~nd 168 2037~C.15' 1OO~LI 2 1 0 25 6 2 189 2037~C.15' lOO,ul 15.4 1.62 119 3 185 2037~C.15' 1OO~LI 9.2 0.99 66 2 4 95 537~C.15' 100~11 44 0 2.33 6 0.3 100 537~C.15' 100~11 29.0 1.43 43 104 537~C.15' 100~1 36.0 1 70 24 0.4 40n7 Primer Competition Counter-SELEX
SELEX Total TotalTemp, % Bound % 5mMEDTA Kd(nM) Round RNA Protein Time. LS-RE EDTA Si~nal/
pmoles pmoles Vol. SitesEluted Bk~nd 155 2037~C.15' 100~11 1.9 0.25 5 2 184 2037~C.15' 100111 26.8 2.92 172 3 117 2037~C.15' 100111 12.9 2.21 78 2 4 93 537~C.15' 100111 46.0 2.43 3 0 2 93 537~C.15' 1OO~LI 37.0 2.00 52 94 537~C.15' 1OO~LI 42.0 2.25 15 0.06 WO g~a703 PCT/U~ r~ ro3 1~5 1û7 a H

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~o ~ r.~ u~ ~ o ,~ o u~ u m oo - u~ r.- u~ r c~ Ln r W096/40703 PCT/U' Q''~31~5 Table 17 2' Fluoro L-selectin SELEXes:
Full Length Transcribed Ligands:
Protein and Lymphocyte Binding Affinity L-selectin# Lyrnphocytes# #
LIGAND SEO ID NO Kd (nM) Kd (nM) LF1508 307 0.5 LF1511 342 0.48 LF1513 321 0.16 4 LF1514 297 0.13 0.8 LF1516 374 1.3*
LF1518 293 0.42 LF1520 339 0.5*
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 25 LF1707 356 0.16*

WO ~"~C703 PCT/U'r''~3'~' Table 17 (Page 2) L-selectin# Lymphocytes# #
LIGAND SEQ ID NO Kd (nM) Kd (nM) 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 LF1815 ' 305 0.18*
LF1816 335 -*
LF1817 294 2.3*

NX280 1.6 3 # Nitrocellulose flter partitioning @ 37~C;
* design~te soluble L-selectin, others LS-Rg;
- inrlic~f~o.s binding was undetectable ## Flow cytometry colllpetiLion @ room temperature;

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fCS J ~ J r~ c r'l rl~l cSl c I c'l ~sl fS fl f r C; fS CJ
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C r- C- C r- r- ~-1 rS ~-1 C, ~-1 rr r_ ~ ~ r CJ C, CJ C. C~ CJ~r~ r ~ ~r~ ~ ~r~ fS ~r~ r , r~
C~ C C1 C C C ~rS ~ rS E ~ i~ C ~ ~
rS I rS fS rS rS rSn~ ~S r~ ~ ~ C, cc I cc c~ c~ cc cr~ c, cJ ~ r, _ C
rS rS rS rS r~ rS C ~ rS ' r~ ~ rS r rS rS rs rs r; rS ~ ~ rs J c , ~ , r~ Cj C g g ,j g r ~ C~' ~ n n r C, C, r C~ r~
7 ~ 7 ~ ~

~r~
~I V
r~
~ ' Ll 7 ,1 " r ,7 . .
57 ~ 1 7 1 7 r~ u7 ~ 7 7 7 1~5 aJ --I i~'7 ~ D r.~7 U'l r.~l r.~l ' ' ' ' r~ D r.~ ~ ~--t~ ~ ~ ~ ~ ~ ~ ~ r--rS ~ ra~ r~7 r~ o ,~
r7 ;Y7r7 r7 ~ rl r7 r PCT/US96'0~ 1~S

Table 20 Dissociation Constants and Specificity of 2'F RNA
Ligands to P-Selectin Kd SLeX Kd Kd SEQID
Ligand (PS-Rg) (IC50) (ES-Rg) (LS-Rg) Tm(oC) NO.
PF37349.5pM >3~M ~3~M 199 PF37718.5 pM 3nM 2.3~M >3~M 53OC 206 PF37851.5 pM 212 PF38074.5 pM 4nM 205 PF38116.5 pM lnM 213 PF38645.5 pM 216 PF38716pM 207 PF38890pM 211 PF39526pM 209 PF39624pM 215 PF39846pM 204 PF40447.5 pM 218 PF41113pM 2nM 214 PF412450 pM 201 PF41663pM 210 PF41769pM 219 PF422172 pM 3nM 202 PF42436.5 pM 200 WO g6/~.a70~ PCT/U~ 5' O ~ ~ ~'1 ~ ~ ~ ~'I N ~'1 ~ ~ r.

u~

~ 1 c C C~
C') C~C~c7 C~ c) C~
U C) C~ C) C) ~ ~ ~) C) ~ ~.
C r rr r C; C r~ C r_ r_ r~ C ~ C 2 _ ~ ~ ~ ~ r_ ~ r~ ~ I ~ ~ ~ 1~ CJ
CcJ ~J _ J _ , C~ , _ ~ J C, J
c~ 'I c~ I c'~ ~'1 c~
~D c~ ~ ' ~c5 ~ ~ - ,~ c~ ''c5 cf~ fc~ C C~
C ~ ~ f~ C, ~ f I
O;Z ~ I C~ I C~l C~l ~ l ~1 ~1 ~I Cll _l C~ C, r ' r --I
I ~ I Cl Cl Cl Cl C~l C)l C71 C~ C~l _'l C~
~e f ~ C C~ J ~ . C, P~ ~ ~I C)l ~ ~I C)l rt C~l C)l ~I C)l _l ~ I Jl ~ ~) ~ r- rt: :)1 ml ::~ 3131 ~ 31 31 ~1 31 ?1_1 -I - ~ ~¦ ~ c ,C
O ~ f r~ C ~ ~ , r~
,~ C, r~ ~ ~ CJ { I _ r~
6 ~ ~ ~ _ ~ _ Ir/ C ~ f~ ' f~
m C C c ~ ,~ J C~ t. ~ r, ~ C 6 ~ C C C~ J ~
~ f~t ~ 'r~ ~
~5 _ _ C, C, ~ C_ J ~ .¢ ~ ~6 J
J ~ C~ C~ ~ f J ~ ~ ~ 3 c r ~ ~ f 1~ r 1~ J lCI, t V ~ -~ ~ ~ c, 8 c ~ c t ~ t. ~ ' _ C 3 IC / t~ ~ ~ ~ J ~ ~ ~ ~ r _ C, .~: ~ r~
~ f~ C cr) C ~ C lc ~ r~ f ~ C~ _ ~) C~ r~ ~
C ~ CJ _ ~ C, I~ C ~ C ~ C :~ VC) J ~:t ~¢ ~ C ~ C, ~ CJ D~ f~ _ t ~ _ C
Z C~ f~ r- ~ C ~ ~ r ~ ~ CJ
~16 cr C ~ r~ CS C~ f~t ~ CJ 16 ~S ~ tCJ ~~ f ~ ~ ~
:1 C~l Ul ~ r31 r~l r~l ¢I r~l C~ r5~ ~1 r5~ ~1 6 ~ _ c a~ ~ 31 -I cll t c)l c~l 31 c~l c)l C)l c~l c~l 31 C~l ' c~ ~ J ~ C~

; ~ ft f~ C) f6 ~ f~ C C~ ~S C C~r ~ C. C ~S ~ r.~ _ ~ ~ ~ tJ Ln ~ U~ C
~15 fl C f~ C' f~ 16 C C~ 7r 6 i I Z
f~ ~ C~ rr ~ 16 ~ ~ cr ~ ~~ I Cl ~61 Cl r~l Cl ~I ft¦ f61 _1 rl Cl ~I r,,l H; H ~ H ' H ~ ~4 ~ f U ~ f r~ ~5~ 5~1 ~Cl Cl Cl Cl ~1 Jl Cl C,q .I ~5, t~ f~ rC ~ S fE C, o t~ f, m ~ cJI l ~ c ~ .
r5 r'' C I ' f~ f~ r ~5 A ~5 f .

r5' t') r O U U U U C) ~ I r5 n5 ~) r n ,, r ~

O 1' m .

a ~ ~ o r r r.~ r r~ r~ ~ Ln ~o r.~l t~ ~ ~ ~ ~D r ~ ~ r r.~ rJ~ r~ r r ~ r~ r~ r~ ~ rJ~ r.~ r.~l r r~ r~ ~ r~ ~
C ~ ~ r~ r~) r~- r~ ~ r~ r~) r~ ~ r~ ~ ~ r~ r~ r~7 ~ r~ ~
C, Ci, ~ ~ ~ C~ G G ~ ~ P~ ~ ~ CL, ~ ~4 ~ ~ ~ ~ CL, :~: o Q. o o r,~ o O O
~rJ~D r r~ o o rr~ o u~ o ~ U~ ~1 ~ Z u~

PCT/U~r'~g '~' Table 22 Dissoci~,l;on Constants and Specificity of Truncated 2'F RNA Ligands to P-Select;I) Kd S LeXKd Kd Tm # SEQID
Ligand(PS-Rg) (IC50) (ES-Rg) (LS-Rg) (oC)Bases NO.
PF373s156 pM 3 nM ~ 3 IlM > 3 IlM 220 PF377s160 pM 2 nM > 3 ~lM ~ 3 ~lM 59OC 38 223 PF377s245 pM 4 nM 42 224 PF383sl10000 pM 25 nM 46 227 PF387sl63 pM 2 nM > 3 ~lM > 3 ~M 46 226 PF398sl178 pM 2 nM > 3 ~lM ~ 3 ~M 39 222 PF416s2150 pM 3 nM 42 228 PF422s11000 pM 8 nM > 3 ~LM > 3 ~lM 44 229 PF377s1B65 pM ~ 3 nM > 3 ~lM > 3 IlM 38 223 PF377slB:SA30 pM 38 223 PF377slF60 pM 3 nM 38 223 PF377sl -5'NH2125 pM 2 nM 41 223 PF377L1220 pM 4 nM ~ 3 IlM > 3 ~lM 35 225 PF377t3'30 pM 2 nM 59 223 PF377M1120 pM > 3 ~uM 38 230 PF377M21700 pM 3 8 2 31 PF377M3900 pM 10 nM >3 ~lM 38 232 PF377M41700 pM 38 233 PF377M560 pM 2 nM > 3 ~LM 38 234 PF377M6250 pM 38 235 CA 0222327~ 1997-12-02 PCT~U~ 3~5 Table 23 2'0Me Substitution of 2'F RNA
Ligands to P-''el~ti"
Purine Unmixed Std. Dev.Mixed Mixed Predicted Actual Position Ratio 40 pM200 pM Pref. Pref.
4 1.07 0.12 0.3 0.4 2'-OH untested 1.00 1.00 0.4 0.7 2'-OH untested 7 1.00 0.13 1.2 1.5 2'-O-Me 2'-O-Me 8 1.00 0.20 2.3 1.3 2'-O-Me 2'-O-Me 1 2 0.83 0.12 0.4 0.5 2'-OH untested 13 0.90 0.17 0.8 0.8 neutral 2'-O-Me 14 0.73 0.15 0.8 0.9 neutral 2'-O-Me 0.63 0.15 0.8 1.3 2'-O-Me 2'-O-Me 16 0.67 0.10 0.5 0.7 neutral untested 18 0.60 0.10 0.7 0.7 neutral 2'-O-Me 21 0.87 0.30 0.5 0.7 neutral 2'-O-Me 22 0.72 0.16 0.7 0.8 neutral 2'-O-Me 24 0.70 0.16 0.6 0.8 neutral 2'-O-Me 27 0.83 0.12 1.3 1.5 2'-O-Me 2'-O-Me 28 0.69 0.09 0.6 1.0 2'-O-Me ?
0.90 0.00 0.8 1.0 neutral ?
31 0.92 0.16 1.2 1.5 2'-O-Me 2'-O-Me 32 1.10 0.06 0.5 0.8 2'-OH untested 34 0.93 0.06 0.7 0.9 2'-OH untested WO 9~/~C703 PCT/U~ 5 ~55 ~ , a~
OD ~ I~ ~ ~ ~

CO

_ . ~D ~ ~ c~7 C~ D c~ O O N O
C O o o O o O O _ O O--O U~
C O

~ o ~ i ~ ~ C~ N -- V N V V V N ~

Z ~ CD a7 ~ o o . o o o c'J cu --O O O--O O O O O O O O O o _ ~ _ c~ O N U7 1~ 1~ U~ C~ C~.l C~.l ~ C~.l c Z ~ ~ ~ o ~:~ CD O U~ a~ _ ~ CD C" ~ O U~-- .
O O O -- -- -- -- -- c~ C~ C'.i N ~ C'~

.~
t t t t t t ~ t ( t t ; t C N g C~l N ID N ~ o o o o o t t _ ~. . -- t t ~ t t t t t m o ~~~~~~_~~~~_~_~
C~J C~l F ~ ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ~ ~
~L ~ N _ _ _ _ J ~ ~.
~ C ~ ~~ ~ 0~ CD O ~o ~ ~ ~----_ _ _ G

U. ~ _ N cr~ a7 ~-- N
C ~1 ~J
O ~ ~ G C ~ C ~ C ~ ~ G

WO 9~'JC703 PCT/US96~'~9'~5 ~ ~ rD r~ o ,~ r.~l r~ n ~D t~ r~ o H O r~ t~ co r~ r~ I r~ 0 r~ r~ co r~ 0 cJ~

. 1 . . . . . . . . . . .
. . . . . . . . . . . . .

. . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .

I ) . 1 1 1 1 1 1 1 1 ~ I

C ~' f'' U f~ f~' C f t ~ C' C CJ f C C C.
f t C, ~ f~ ~ fS ~ C~ f C. fS
C f t ~ fI ~t ~) f~ Cr ~ f~ C r ~ I r C, fS ~ _ C f't C C f t _ ' r t ~ CJ _ _ t C, C ~t:. C U f _ ~ ~ ~
f ~ f t f - J -- f r ~ fCS _ ~ ~ f C' f _ ~ CJ _ ~ f C Z
f '; J fl 3 C~ J t C, _ C) f ~ f~ C, J C~ _ ~ fS f~ ~
C, 'r' C, r' fS '~ crJ _ CJ C~ Z
C fS fS l ~_ CJ fS fS _ t crJ crJ Cl N C c~ cs c , cC'J I J _ f ~ ~ C;~
;J crJ f~ Ct C) CJ Cl ~D f ~ Cl f C. ~ f' _ C fS CJ fl;
t~f ~ Cl f f t ~ C, r~ C, ~ C~ C) f ~ ~ C _ f f~ C) ~4f f ~ ~ fS _ C~; f CJ f r~ fS~
~ t ~ ~S ~ _ f ~ -- f U crJ
f t ' _ 3 f;. ~ f fS
~ f ~ crJ '' J -- : f~ crJ C) U~fS Cr ~ ~; ~ C ~ ~ &
N: C, C f~ f , ~' ~ ~ _ f f ~1 f~ f~ f J ' f C C ~ _ ; ~ f ~7 _ ~. c f _ C ~
_J C _ r f f ~ C. _ C f J

, r ~ C C ~ ~_ C f C C, C
E~l f.~ J ~ ~ _ f~ ~ r c _~, J _ C, C CJ f~ _.r _ _ ~ ~; '~ f t f~ C~ . f C _ ~t~ f J ~ f ~ _ t CCJ ~ J Cl f~ C C. C -t - --f~ C' _ ~ C~ f' C, _ _~
f S ~ t ~ ~ f _ ~ ~
fS 'f~ fS ~S ~ f _ C Z ~ , fS C~) ~ r' ~ f C Z 'J
f~ C. ~ ~ f' C Z t ~_ ~ ~, n, . 1 ~ 1 11 1 1 lZ

~D
r~ n~ r~
r~ 1 1 1~ 1 1 1 1 ~ 1 1 1 1 cn 1 1 ~ 1 1 ~ ~ ~ cn ~ ~

r~ ~ r.~ r.~ r ~ t~ r~ ~n t~ a~
r~ ~ ~ ~ tn ~ ~n u~
~I rr~ rr~ r~l r~ r~ r~l r~ r~l ~ ~ ~P ~r ~
~S~ ~S~S~S~SI~ ~ f S ~S~S ~ ~S

CA 0222327~ 1997-12-02 PCT/U~ "03 155 WO ~ 703 Table 26 I)issoci<-lion Constants and Speci~ioil~ of 2'NH2 RNA Ligands to P-Selecli-, Kd Kd SLeX Kd Kd SEQ ID
Ligand (PS-Rg) (40C)(IC50) (ES-Rg) (LS-Rg) N0.
PA3012.5nM 264 PA3050.21 pM 265 PA3090.656 pM 266 PA3155nM 267 PA3182nM 268 PA31911nM 269 PA3204.5nM 270 PA3218nM 271 PA325>10nM 259 PA32713.5 nM 260 PA3283nM 256 PA3294nM 273 PA3300.237 nM ~ 274 PA33510.5 nM 276 PA33615nM 277 PA3374.5nM 257 PA33857nM 278 PA33913.5 nM 279 PA3410.44 nM 3nM 251 PA3424nM 280 PA3500.06-nM 0.01 nM 2nM 375 nM >3~M 252 PA3512nM 282 PA3526nM 283 PA3539nM 284 PA3545nM 285 PA44750nM 286 PA4485nM 258 PA4638nM 287 PA465>50nM 288 PA4660.43 nM 253 PA46724nM 289 PA4730.36 nM 254 PA4770.57 nM 255 CA 0222327~ 1997-12-02 WO 96/40703 PCT/U~3G~ S

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: PARMA, et al.
(ii) TITLE OF INVENTION: HIGH A ~lNlLY NUCLEIC ACID
LIGANDS TO LECTINS
(iii) NUMBER OF SEQUENCES: 390 (iv) CORRESPON~N~ ADDRESS:
(A) ADDRESSEE: Swanson & Bratschun, L.L.C.
(B) STREET: 8400 E. Prentice Avenue, Suite 200 (C) CITY: Englewood (D) STATE: Colorado (E) C~UN1KY: USA
(F) ZIP: 80111 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3 1/2 diskette, 1.44 MB
(B) COMPUTER: IBM pc compatible (C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WordPerfect 6.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/09455 (B) FILING DATE: 05 JUNE 1996 (C) CLASSIFICATION:
(vii)PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/479,724 (B) FILING DATE: 07-JUNE-1995 (vii)PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/472,256 (B) FILING DATE: 07-JUNE-1995 (vii)PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/472,255 (B) FILING DATE: 07-JUNE-1995 (vii)PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/477,829 (B) FILING DATE: 07-JUNE-1995 (viii)ATTORNEY/AGENT INFORMATION:
(A) NAME: Barry J. Swanson (B) REGISTRATION NUMBER: 33,215 (C) R~K~N~/DOCKET NUMBER: NEX40C/PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 793-3333 (B) TELEFAX: (303) 793-3433 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GGGAAAAGCG AAUCAUACAC AAGANNNNNN ~NNN~NN NN~NNNNN~N 50 CA 0222327~ l997-l2-02 W O 96/40703 PCT/U~6/~l5' N NN'NN~ NN~ N N~NN~NNNN NNNNGCUCCG CCAGAGACCA ACCGAGAA 98 (2) INFORMATION FOR SEQ ID NO:2:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) sTRpNn~n-N~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
W CUCGG W G ~u~u~uGGCG GAGC 24 (2) INFORMATION FOR SEQ ID NO:4:
(i) ~u~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) S~U~N~ DESCRIPTION: SEQ ID NO:4:

(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
~ (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ 1997-12-02 W O ~ D703 PCT/U~ 5 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGGAAAAGCG AAUCAUACAC AAGACAGGCA CUGA~AACUC GGCGGGAACG 50 ~2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9l base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGGAAAAGCG AAUCAUACAC AAGAAGUCUG GCCA~AGACU CGGCGGGAAC 50 GUAAAACGGC CAGAA W GCU CCGCCAGAGA CCAACCGAGA A 9l (2) INFORMATION FOR SEQ ID NO:7:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STR~NnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRPNnRnNR~5 single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All Cls are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GGCUCGGCGG GAACGA~AUC UGCUCCGCCA GAGACCAACC GAGAA 95 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ 1997-12-02 W O 9~/~C703 PCT/U~5~ 03 l5 (D) TOPOLOGY linear (i1) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE:
(D) OTHER INFORMATION: A11 UIS are 2'-NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO 9 GGGA~AAGCG AAUCAUACAC AAGA W GGGC AGGCAGAGCG AGACCGGGGG 50 CUCGGCGGGA ACGGAACAGG AAUGCUCCGC CAGAGACCAA CCGAGA~ 9 7 (2) INFORMATION FOR SEQ ID NO:10:
(i) ~U~N-~ CHARACTERISTICS
(A) LENGTH 97 base pairs (B) TypE nucleic acid (C) STRA~N~SS single (D) TOPOLOGY 1 inear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OL~R INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE:
(D) OTHER INFORMATION A11 UIS are 2' -NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:10:
GGGA~AAGCG AAUCAUACAC AAGA~AGGGA UGGGAUUGGG ACGAGCGGCC 50 AAGACUCGGC GGGAACGAAG GGUGCUCCGC CAGAGACCA~ CCGAGAA 9 7 (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 96 base pairs (B) TYPE nucleic acid (C) STRA~N~SS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 UIS are 2' -NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:11:
GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGA~A GUGUCAUGGU 50 (2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTX 98 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2' -NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 U~S are 2' -NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:12:

CA 0222327~ l997-l2-02 WO 9"~703 PCTAUS9~l~g1'~

~2) INFORMATION FOR SEQ ID NO:13:
(i) s~Qu~ CHARACTERISTICS: .
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid ~C) STRAN~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGGA~AAGCG AAUCAUACAC AAGAAAGGGA UGGGA W GGG ACGAGCGGCC 50 (2) INFORMATION FOR SEQ ID NO:14:
(i) s~Qu~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~ ~ DESCRIPTION: SEQ ID NO:14:
GGGAAAAGCG AAUCAUACAC AAGACUCGGC GGGAACGAAG u~u~u~AGuA 50 (2) INFORMAUION FOR SEQ ID NO ls (i) ~yu~ CHARACTERISTICS:
(A) LENGTH: 100 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

(2) INFORMATION FOR SEQ ID NO:16:
(i) ~u~N-~ CHARACTERISTICS:
(A) LENGTH: 103 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATuKE:

CA 0222327~ l997-l2-02 W O 9~ 703 PCTAUS9~S'~S

(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GGGA~AAGCG AAUCAUACAC AAGACUCGGC GGGAAUCGUA AUGUGGAUGA 50 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-N~2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) ~U~N~ DESCRIPTION: SEQ ID NO:17:

(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANI~:l)N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GGGAAAAGCG AAUCAUACAC AAGACGGCUG u~u~u~uAG CGUCAUAGUA 50 GGA~uC~u~A CGAACCAAGG CGCUCCGCCA GAGACCAACC GAGAA 95 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid CA 0222327~ l997-l2-02 W O g~'~Cl~3 PCT/U~ '0g15' (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GGGAA~AGCG AAUCAUACAC AAGACGAUGC GAGGCAAGAA AUGGAGUCGU 50 (2) INFORMATION FOR SEQ ID NO:21:
U~N-~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
~ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:21:

(2) INFORMATION FOR SEQ ID NO:22:
( i ) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:22:

GGGCAAUGGA ~u-~u~ACGA ACCGCUCCGC CAGAGACCAA CCGAGAA 97 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

CA 0222327~ l997-l2-02 W O 9G/~C703 PCT/U'~'0~1~5 (2) INFORMATION FOR SEQ ID NO:24:
(i) ~yu~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANnRnNR~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-N~2 uracil (xi) ~:yu~N~ DESCRIPTION: SEQ ID NO:24:

(2) INFORMATION FOR SEQ ID NO:25:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All Cls are 2~-NH2 cytosine (ix) FEAlUKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:25:
GGGAAAAGCG AAUCAUACAC AAGAGAUACA GCGCGG~u~u AAAGACCUUG 50 (2) INFORMATION FOR SEQ ID NO:26:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDN_SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yU~N~ DESCRIPTION: SEQ ID NO:26:

(2) INFORMATION FOR SEQ ID NO:27:
(i) S~yu~:N~ CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ 1997-12-02 WO 96/40703 PCT/US~ 155 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GGGAAAAGCG AAUCAUACAC AAGACA~ACC UGCAGUCGCG CGGUGA~ACC 50 (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

(2) INFORMATION FOR SEQ ID NO:29:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~n~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAlUKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single S~E~ (RULE 9t) CA 0222327~ l997-l2-02 WO~~'~C1~3 PCT/U' 5'1'~}S''~

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

(2) INFORMATION FOR SEQ ID NO:32:
(i) s~:Qu~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All Cls are 2'-NH2 cytosine (iX) FEATuKE~
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

G~uu~u~AGG A W CGCCA W AGGCGCUCCG CCAGAGACCA ACCGAGAA 98 (2) INFORMATION FOR SEQ ID NO:33:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANnRnN~.~S: single (D) TOPOLOGY: linear (il) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~ DESCRIPTION: SEQ ID NO:33:

(2) INFORMATION FOR SEQ ID NO:34:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

Htl~llt~l~ SHEET(RULE91) CA 0222327~ 1997-12-02 WO g"~703 PCT/US9~1~5 ~r (2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRA~ N~:~S: single ~ D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) ~U~N~' DESCRIPTION: SEQ ID NO:35:

(2) INFORMATION FOR SEQ ID NO:36:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~cs single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) ~yU~N~ DESCRIPTION: SEQ ID NO:36:

(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) O'l~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STR~N~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 WO 9~'~C70~ PCTAUS96/0945 (D) OTHER-INFORMATION: All U's are 2'-NH2 uracil (xi) S~yU~N~ DESCRIPTION: SEQ ID NO:38:
GGGA~AAGCG AAUCAUACAC AAGAG W GGU GCGAGCUGGG GCGGCGAGAA 50 (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) S~U~:N~'~ DESCRIPTION: SEQ ID NO:39:
GGGA~AAGCG AAUCAUACAC AAGACUGGCA AGRAGUGCGU GAGGGUACGU 50 (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GGGA~AAGCG AAUCAUACAC AAGA W GGUC GUACUGGACA GAGCCGUGGU 50 AGAGGGA W G GGACA~AGUG UCAGCUCCGC CAGAGACCAA CCGAGAA 97 (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C~s are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:41:
GGGA~AAGCG AAUCAUACAC AAGAUGUGAG A~AGUGGCCA ACU WAGGAC 50 (2) INFORMATION FOR SEQ ID NO:42:
( i ) S ~:Qu~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ 1997-12-02 W O 96/40703 PCTAU596'09'~r (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~ DESCRIPTION: SEQ ID NO:42:
GGGA~AAGCG AAUCAUACAC AAGACAGGCA GAu~u~u~uG A~uu~uCGG 50 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:43:
GGGA~AAGCG AAUCAUACAC AAGAUGUGAU UAGGCAG W G CAGCCGCCGU 50 (2) INFORMATION FOR SEQ ID NO:44:
(i) S~:~u~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~CS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
GGGA~AAGCG AAUCAUACAC AAGAUGCCGG UGGAAAGGCG GGUAGGUGAC 50 (2~ INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
GGGA~AAGCG AAUCAUACAC AAGAGAGGUG RAUGGGAGAG UGGAGCCCGG 50 CA 0222327~ l997-l2-02 W O 9.'~D703 PCT/U~ '091~5 (2) INFORMATION FOR SEQ ID NO:46:
~Uu~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) ST~ Nn~nN~.~s single ' (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~N~ DESCRIPTION: SEQ ID NO:46:
GGGA~AAGCG AAUCAUACAC AAGAGUCAUG CUGUGGCUGA ACAUACUGGU 50 GA~AG W CAG UAGGGUGGAU ACAGCUCCGC CAGAGACCAA CCGAGAA 97 (2) INFORMATION FOR SEQ ID NO:47:
( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRAN~ )N~ ~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
GGGA~AAGCG AAUCAUACAC AAGACCGGGG AUGGUGAGUC GGGCAGUGUG 50 (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STR}~Nn~:nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
~ (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 WO 96/40703 PCT/U' 36~'0~ ''5 (D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

(2) INFORMATION FOR SEQ ID NO:50:
(i) S~QU~N-~ CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) S~yu~N~ DESCRIPTION: SEQ ID NO:50:

(2) INFORMATION FOR SEQ ID NO:51:
(i) S~yu~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NHz cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
GGGA~AAGCG AAUCAUACAC AAGACCCGCA CAUAAUGUAG GGAACAAUGU 50 (2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All Uls are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ l997-l2-02 W O ~5'~D703 PCT/U~3G~'0~155 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:54:
GGGA~AAGCG AAUCAUACAC AAGACGG W G CUGAACAGAA CGUGAGUC W 50 (2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlu~E:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAlu~E:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xl) SEQUENCE DESCRIPTION: SEQ ID NO:55:

(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlUKE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~ DESCRIPTION: SEQ ID NO:56:
AAGACUCGGC GGGAACGA~A 20 (2) INFORMATION FOR SEQ ID NO:57:

CA 0222327~ l997-l2-02 W O ~G"~703 PCT/U',G~ 5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) sTR~NnRn-NR~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:57:
GG2~uC~u~A CGAACC 16 (2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STR~NnRnNRSS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

(2) INFORMATION FOR SEQ ID NO:59:
U ~:N~ CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) sTR~NnRn-NRss single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~u~ DESCRIPTION: SEQ ID NO:59:

(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
U~wNlNNNAG U~N~NNNNNN UAGACGUCGG UGGACNNNGC GG 42 CA 0222327~ 1997-12-02 W O ~ 703 PCT/U~,~.'0~ ~r (2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~QU~N~: DESCRIPTION: SEQ ID NO:61:
GG~N-NN~uGA CYCGRGGAYU C 21 ~2) INFORMATION FOR SEQ ID NO:62:
yU~N~ CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yU~N-~ DESCRIPTION: SEQ ID NO:62:
UGANCNNACU G~u~N-NN~N~ NAG 23 (2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:

(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
GGGAGGACGA UGCGGNNNNN ~-N~N~N NNNNNNNlNNN NN~n~NNN-N 50 CA 0222327~ l997-l2-02 W O 9~'~C703 PCT/U'jc~'0 NNl~NNCAGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:65:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STR~-Nn~nN~S: single (D) TOPOLOGY: linear ., (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~yU~N~ DESCRIPTION: SEQ ID NO:65:

(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) sTRpNnRnN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:66:
TCGGGCGAGT C~lC~lG 17 (2) INFORMATION FOR SEQ ID NO:67:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAN~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
GGGAGGACGA UGCGGCGCGU AUGUGUGA~A GCGUGUGCAC GGAGGCGUCU 50 (2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) ST~n~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 W O ~!S~703PCT/U'5~05'~$

(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:68:
GGGAGGACGA UGCGGGGCAU Wu~u~AAUA GCUGAUCCCA CAGGUAACAA 50 (2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) sTRpNn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlu~E:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

(2) INFORMATION FOR SEQ ID NO:70:
U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPNn~nNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:

(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:

(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ l997-l2-02 WO ~ 7~3 PCT~U59G~ 155 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
GGGAGGACGA UGCGG W GAG Au~u~u~AGU ACAAGCUCAA AAu~CC~uu~ 50 (2) INFORMATION FOR SEQ ID NO:73:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:73:
GGGAGGACGA UGCGGUAGAG GUAGUAUGUG UGGGAGAUGA A~AUACUGUG 50 GA~AGCAGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:74:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
GGGAGGACGA UGCGGA~AGU UAUGAGUCCG UAUAUCAAGG UCGACAUGUG 50 (2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
GGGAGGACGA UGCGGCACGA A~AACCCGAA W GGGUCGCC CAUAAGGAUG 50 CA 0222327~ l997-l2-02 W O gC_C703 PCT/U~3CI~.

(2) INFORMATION FOR SEQ ID NO:76:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nNR~S: single ' (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTn~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~N~: DESCRIPTION: SEQ ID NO:76:

GA~ACCAGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~:N~ DESCRIPTION: SEQ ID NO:77:

(2) INFORMATION FOR SEQ ID NO:78:
u~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Oln~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R lN~O~ATION: All U's are 2'-NH2 uracil (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:78:

(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 W O ~C/~703PCT/U~r~ S

(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) S~:~U~N-~ DESCRIPTION: SEQ ID NO:79:

(2) INFORMATION FOR SEQ ID NO:80:
( i ) S~U~N-~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~QU~N~ DESCRIPTION: SEQ ID NO:80:

(2) INFORMATION FOR SEQ ID NO:81:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) S~QU~N-~ DESCRIPTION: SEQ ID NO:81:

(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nl7~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
GGGAGGACGA UGCGGGAACA UGAAGUAAUC A~AGUCGUAC CAAUAUACAG 50 (2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRAWDEDNESS: single CA 0222327~ l997-l2-02 W O ~ t70~ PCT/U~ '03'~5 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine - (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yU~N~ DESCRIPTION: SEQ ID NO:83:

(2) lN~G~ATION FOR SEQ ID NO:84:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Or~R lN~O~ATION: All U's are 2'-NH2 uracil (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:84:

CCAAAACAGA CGA u~GCCC GA 72 (2) lN~ORI~TION FOR SEQ ID NO:85:
(i) ~yu~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRpNn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER lN~O~ATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-~H2 uracil (xi) ~:yU~N~ DESCRIPTION: SEQ ID NO:85:
GGGAGGACGA UGCGGGAACA UGAAGUAA~A GUCUGAG W A GUA~A W ACA 50 (2) INFORMATION FOR SEQ ID NO:86:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
- (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NHz cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yU~N-~ DESCRIPTION: SEQ ID NO:86:

CA 0222327~ 1997-12-02 WO 96/40703 PCT/U'~6/03~5' (2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:CS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Or~R INFORMATION: All U's are 2'-NH2 uracil (Xi) ~U~N~: DESCRIPTION: SEQ ID NO:87:

(2) INFORMATION FOR SEQ ID NO:88:
(i) S~u~ : CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:

(2) INFORMATION FOR SEQ ID NO:89:
( i ) S~UU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) S~U~N~: DESCRIPTION: SEQ ID NO:89:
GGGAGGACGA UGCGGUAACA UAAAGUAGCG C~u~u~u~AG AGGAAGUGCC 50 (2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ 1997-12-02 W O 9~'~D70~ PCT/U~5~'~g1r5 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
GGGAGGACGA UGCGGAUAGA ACCGCAAGGA UAACCUCGAC C~u~Gu~AAC 50 (2) INFORMATION FOR SEQ ID NO:91:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
~ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
GGGAGGACGA UGCGGUAAGA ACCGCUAGCG CACGAUCA~A CAAAGAGA~A 50 CA~ACAGACG ACUCGCCCGA 70 (2) INFORMATION FOR SEQ ID NO:92:
(i) S~N'~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER lNru~ ~TION: All C~s are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
GGGAGGACGA UGCG~uu W C UCCAAGAACY GAGCGAAUAA ACSACCGGAS 50 (2) INFORMATION FOR SEQ ID NO:93:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) S~NUE DESCRIPTION: SEQ ID NO:93:

(2) INFORMATION FOR SEQ ID NO:94:
(i) S~U~:NU~' CH~RACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) sTRA~n~nN~.~s single CA 0222327~ l997-l2-02 W O ~ 703 PCT/U~r~'051'~

(D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER lN~O~ATION: All U's are 2'-N-H2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:

AUCCCCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
GGGAGGACGA UGCGGUCCCA GA~u~CC~u~ AUGCGAAGAA UCCA W AGUA 50 CCAGACAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO:96:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAlu~E:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
GGGAGGACGA UGCGGGAUGU AAAUGACA~A UGAACCUCGA AAGA W GCAC 50 (2) INFORMATION FOR SEQ ID NO:97:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:

CA 0222327~ l997-l2-02 W O 96/40703 PCT/U'96'~9''' (2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) sTRpNn~n~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEALuKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:

(2) INFORMATION FOR SEQ ID NO:99:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~u~N~ DESCRIPTION: SEQ ID NO:99:

(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~U~N~ DESCRIPTION: SEQ ID NO:100:
GGGAGGACGA UGCGGCA~AA GG W GACGUA GCGAAGCUCU CAAAAUGGUC 50 (2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 W O 96/40703PCT/U~9~'0~5C5 (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~ DESCRIPTION: SEQ ID NO:101:
GGGAGGACGA UGCGGAAGUG AAGCUA~AGC GGAGGGCCAU UCA~uuu~C 50 ~2) INFORMATION FOR SEQ ID NO:102:
u~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
GGGAGGACGA UGCGGAAGUG AAGCUA~AGS GGAGGGCCAC UCAGA~ACGC 50 (2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:

~2) INFORMATION FOR SEQ ID NO:104:
u~ CHARACTERISTICS:
(A) LENGTH: 67 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEALu~E:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATuKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:

(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~nRnNR~S: single CA 0222327~ l997-l2-02 W O 9~ 703 PCT/U~ S~5 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
' (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:

(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:

(2) INFORMATION FOR SEQ ID NO:107:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:107:
GGGAGGACGA UGCGGUCAGA ACUCUGCCGC UGUAGACA~A GAGGAGC W A 50 (2) INFORMATION FOR SEQ ID NO:108:
(1) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
- (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytoslne (ix) FEAlU~E:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:

CA 0222327~ l997-l2-02 W O 9''~0703 PCTAUS9~'~g155 (2) INFORMATION FOR SEQ ID NO:109:
(i) S~:Qu~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) S~u~N~ DESCRIPTION: SEQ ID NO:109:

(2) INFORMATION FOR SEQ ID NO:110:
(i) ~yuk~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
GGGAGGACGA UGCGGGAUGC AGCAACCUGA A~ACGGCGUC CACAGGUAAU 50 (2) INFORMATION FOR SEQ ID NO:lll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(lx) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lll:

(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:

CA 0222327~ 1997-12-02 WO 9G/~703 PCTrU59G/091~' (D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:112:

(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) O'l~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:

(2) INFORMATION FOR SEQ ID NO:114:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:

(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:

(2) INFORMATION FOR SEQ ID NO:116:
U~N-~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ l997-l2-02 W O g~'~D703 PCT~US~ 9155 (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2' -NH2 cytosine (iX) FEATURE:
(D) OTHER INFORMATION A11 UIS are 2' -NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:116:

(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 67 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 UIS are 2'-NH2 uracil (Xi) ~U~N-~ DESCRIPTION SEQ ID NO:117:
GGGAGGACGA UGCGGUCCAG AGCGUGAAGA UCAACGUCCC G~N~UCGAAG 50 (2) INFORMATION FOR SEQ ID NO :118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 8 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY 1 inear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 U~S are 2' -NH2 uracil (Xi) S~U~N-~ DESCRIPTION SEQ ID NO:118:
AU~U~U~A 8 (2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 15 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY 1 inear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 U~S are 2'-NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:119:

(2) INFORMATION FOR SEQ ID NO 120:
(i) SEQUENCE CHARACTERISTICS

CA 0222327~ l997-l2-02 W O g~ 703 PCT/U'3~/09~5' (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:120:

(2) INFORMATION FOR SEQ ID NO:121:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:121:

(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STR~NI IN I )N'~ s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi ) ~Q~N~ DESCRIPTION: SEQ ID NO:122:

(2) INFORMATION FOR SEQ ID NO:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlUKE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:123:

(2) INFORMATION FOR SEQ ID NO:124:

CA 0222327~ l997-l2-02 W O ~ C703 PCT~Us96~'~5155 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:

(2) INFORMATION FOR SEQ ID NO:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:

(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) sTRANn~NF~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~u~N-~: DESCRIPTION: SEQ ID NO:126:
CTACCTACGA TCTGACTAGC N~NNNN~NNN NNNN~NN~NN ~N~NNNNNN 50 NNNN~NN~ GCTTACTCTC ATGTAGTTCC 80 (2) INFORMATION FOR SEQ ID NO:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:

(2) INFORMATION FOR SEQ ID NO:128:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA

CA 0222327~ l997-l2-02 WO 96/40703 PCT~uS9~'~3155 (xi) FEATURE:
(D) OTHER INFORMATION: N AT POSITION 2 AND 4 IS BIOTIN
(xi) ~yu~ DESCRIPTION: SEQ ID NO:128:

(2) INFORMATION FOR SEQ ID NO:129:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~N~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~b: DESCRIPTION: SEQ ID NO:129:

GTA~ACACTT GCTTACTCTC ATGTAGTTCC 80 (2) INFORMATION FOR SEQ ID NO:130:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRaN~ l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~U~N~ DESCRIPTION: SEQ ID NO:130:

GTA~AATCAG GCTTACTCTC ATGTAGTTCC 80 (2) INFORMATION FOR SEQ ID NO:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:131:
CTACCTACGA TCTGACTAGC GGCATCCCTG AGTCATTACA AG~ll~LlAA 50 (2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~NI)~:l)N~:cs: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~:yu~N~ DESCRIPTION: SEQ ID NO:132:

(2j INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0222327~ l997-l2-02 W O ~ 703 PCT/U','~4 (ii) MOLECULAR TYPE: DNA
(xi) S~U~N~ DESCRIPTION: SEQ ID NO:133:
CTACCTACGA TCTGACTAGC CAC~LLl-~AA GGGGTTACAC GA~ACGATTC 50 (2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANv~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~Uu~N~ DESCRIPTION: SEQ ID NO:134:
CTACCTACGA TCTGACTAGC CGGACATGAG CGTTACAAGG TGCTA~ACGT 50 (2) INFORMATION FOR SEQ ID NO:135:
( i ) S~UU~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:

(2) INFORMATION FOR SEQ ID NO:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:

(2) INFORMATION FOR SEQ ID NO:137:
( i ) ~QU~N-~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:

(2) INFORMATION FOR SEQ ID NO:138:
(i) s~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear CA 0222327~ 1997-12-02 W O 9F'~0703 PCT/U'~C/~9'~5 (il) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:

(2) INFORMATION FOR SEQ ID NO:139:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~YU~N~ DESCRIPTION: SEQ ID NO:139:

(2) INFORMATION FOR SEQ ID NO:140:
yU~N~: CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~CS single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:140:

AL~L~LG~lG CTTACTCTCA TGTAGTTCC 79 (2) INFORMATION FOR SEQ ID NO:141:
Qu~N~: CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:

(2) INFORMATION FOR SEQ ID NO:142:
(i) ~yu~:N~: CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (il) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:

(2) INFORMATION FOR SEQ ID NO:143:
(i) S~yU~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid ~ (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0222327~ l997-l2-02 W O 96/40703PCT~USg6~5~5 (ii) MOLECULAR TYPE: DNA
(xi) S~:YU~N-~ DESCRIPTION: SEQ ID NO:143:

TAAl~lG~lG CTTACTCTCA TGTAGTTCC 79 (2) INFORMATION FOR SEQ ID NO:144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) sTRpNn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:144:

(2) INFORMATION FOR SEQ ID NO:145:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:

(2) INFORMATION FOR SEQ ID NO:146:
(i) S~:QU~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~Qu~ DESCRIPTION: SEQ ID NO:146:

(2) INFORMATION FOR SEQ ID NO:147:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:

(2) INFORMATION FOR SEQ ID NO:148:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRPNDEDNESS: single (D) TOPOLOGY: linear CA 0222327~ l997-l2-02 W O g~'~D703 PCT/U'~'~S1~5 (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:

(2) INFORMATION FOR SEQ ID NO:149:
(i) S~u~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~u~N~: DESCRIPTION: SEQ ID NO:149:

(2) INFORMATION FOR SEQ ID NO:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:150:

(2) INFORMATION FOR SEQ ID NO:151:
u~ CHARACTERISTICS:
(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:

(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:
CTACCTACGA TCTGACTAGC CGGCTATACN NGGTGCTA~A CGCAGAGACT 50 -(2) INFORMATION FOR SEQ ID NO:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRAN~vN~SS: single (D) TOPOLOGY: linear CA 0222327~ l997-l2-02 W O 96/40703 PCTrUS96/09455 (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:

(2) INFORMATION FOR SEQ ID NO:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRPNl,~l,N~sS single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:154:

(2) INFORMATION FOR SEQ ID NO:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~Nl~ sS single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:

(2) INFORMATION FOR SEQ ID NO:156:
(i) ~u~: CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:

(2) INFORMATION FOR SEQ ID NO:157:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:

(2) INFORMATION FOR SEQ ID NO:158:
U~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~.SS: single (D) TOPOLOGY: linear CA 0222327~ 1997-12-02 W O 9f'~0703PCT/U~~'0S'~' (ii) MOLECULAR TYPE: DNA
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:158:

(2) INFORMATION FOR SEQ ID NO:159:
U~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:
CTACCTACGA TCTGACTAGC GCAACGTGGC CCCGLllAGC TCATTTGACC 50 (2) INFORMATION FOR SEQ ID NO:160:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~-~S: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: DNA
(xi) S~U~N~ DESCRIPTION: SEQ ID NO:160:

TGG~L~l~LG CTTACTCTCA TGTAGTTCC 79 (2) INFORMATION FOR SEQ ID NO:161:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:

(2) INFORMATION FOR SEQ ID NO:162:
(i) ~OU~:N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:162:

(2) INFORMATION FOR SEQ ID NO:163:
(i) ~Q~:N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear CA 0222327~ 1997-12-02 W O 9"~703 PCT/U~''091'~

(ii) MOLECULAR TYPE: DNA
(xl) S~U~N~ DESCRIPTION: SEQ ID NO:163:

(2) INFORMATION FOR SEQ ID NO:164:
( i ) S~:QU~N~'~ CHARACTERISTICS:
(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) STRp~n~nN~ss single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(Xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:164:
CTACCTACGA TCTGACTAGC ~L~LlAC CTCATGTAGT TCCAAGCTTG 50 (2) INFORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:165:
CTACCTACGA TCTGACTAGC AGC~Ll~lAC GGGGTTACAC ACAACGATTT 50 (2) INFORMATION FOR SEQ ID NO:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) sTRpNn~nN~ss single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yu~N~ DESCRIPTION: SEQ ID NO:166:

(2) INFORMATION FOR SEQ ID NO:167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 base pairs (B) TYPE: nucleic acid (C) STRPN~ SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:

(2) INFORMATION FOR SEQ ID NO:168:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 0222327~ 1997-12-02 W O 96/40703 PCT/U~r'09'-S

(ii) MOLECULAR TYPE:DNA
(Xi) S~QU~N~ DESCRIPTION: SEQ ID NO:168 CTACCTACGA TCTGACTAGC CGA1C~1~LG TCATGCTACC TACGATCTGA 50 (2) INFORMATION FOR SEQ ID NO:169:
(i) S~U~ CHARACTERISTICS
(A) LENGTH 80 base pairs (B) TYPE nucleic acid (C) STRANV~VN~SS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE DNA
(Xi) S~Q~N~ DESCRIPTION SEQ ID NO 169 (2) INFORMATION FOR SEQ ID NO:170:
(i) ~U~N~ CHARACTERISTICS
(A) LENGTH 80 base pairs (B) TYPE: nucleic acid (C) STRA~n~nN~S: single (D) TOPOLOGY linear (ii) MOLECULAR TYPE DNA
(Xi) ~YU~N~ DESCRIPTION SEQ ID NO:170:

(2) INFORMATION FOR SEQ ID NO:171:
U~N~ CHARACTERISTICS:
(A) LENGTH 79 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY 1 inear (ii) MOLECULAR TYPE DNA
(Xi) S~U~N~: DESCRIPTION SEQ ID NO 171 (2) INFORMATION FOR SEQ ID NO:172:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 80 base pairs (B) TYPE nucleic acid (C) STRPNI)~I)N~.~S single (D) TOPOLOGY linear (ii) MOLECULAR TYPE DNA
(Xi) S~U~N~ DESCRIPTION SEQ ID NO:172:

(2) INFORMATION FOR SEQ ID NO:173:
( i ) S~U~N~ CHARACTERISTICS
(A) LENGTH 90 base pairs (B) TYPE nucleic acid - (C) STRANDEDNESS single (D) TOPOLOGY 1 inear CA 0222327~ l997-l2-02 W O 9G/~703PCTAUS96/09455 (ii) MOLECULAR TYPE: DNA
(xi) S~u~ DESCRIPTION: SEQ ID NO:173:

(2) INFORMATION FOR SEQ ID NO:174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANnRnNF~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:174:

(2) INFORMATION FOR SEQ ID NO:175:
U~N~ CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) sTRaNn~nNR~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:

(2) INFORMATION FOR SEQ ID NO:176:
(i) S~Qu~ CHARACTERISTICS:
(A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:

GACGAAATGA ~l~l~ll~lG GCTTACTCTC ATGTAGTTCC 90 (2) INFORMATION FOR SEQ ID NO:177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:

(2) INFORMATION FOR SEQ ID NO:178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRaNn~nN~S: single (D) TOPOLOGY: linear CA 0222327~ l997-l2-02 W O g~'~D703 PCT/U'9G~'~915 (ii) MOLECULAR TYPE: DNA
txi) ~QU~N~ DESCRIPTION: SEQ ID NO:178:

(2) INFORMATION FOR SEQ ID NO:179:
(i) ~yU~N~: CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:
CTACCTACGA TCTGACTAGC ATGCCCAGTT CAAG~LL~lG ACCGA~ATGA 50 Gll-lG GCTTACTCTC ATGTAGTTCC 80 (2) INFORMATION FOR SEQ ID NO:180:
Qu~N~ CHARACTERISTICS:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:
CTACCTACGA TCTGACTAGC GCAGCGTGGC C~L~llLAGC TCATTTGACC 50 (2) INFORMATION FOR SEQ ID NO:181:
yU~N~ CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~yU~:N~: DESCRIPTION: SEQ ID NO:181:

(2) INFORMATION FOR SEQ ID NO:182:
(i) ~QD~N~ CHARACTERISTICS:
(A) LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~QU~N~ DESCRIPTION: SEQ ID NO:182:

(2) INFORMATION FOR SEQ ID NO:183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:

CA 0222327~ l997-l2-02 W O 9~ 703PCT/U~ 5''~

(2) INFORMATION FOR SEQ ID NO:184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~U~N~ DESCRIPTION: SEQ ID NO:184:

(2) INFORMATION FOR SEQ ID NO:185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~U~N-~ DESCRIPTION: SEQ ID NO:185:
TAGCCAAGGT AACCAGTACA AGGTGCTA~A CGTAATGGCT TCGGCTTAC 49 (2) INFORMATION FOR SEQ ID NO:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STR~N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:186:

(2) INFORMATION FOR SEQ ID NO:187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:
CCAGTACAAG GTGCTA~ACG TAATGG 26 (2) INFORMATION FOR SEQ ID NO:188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:
CGCGGTAACC AGTACAAGGT GCTA~ACGTA ATGGCGCG 38 (2) INFORMATION FOR SEQ ID NO:189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs CA 0222327~ 1997-12-02 W O g~'4G703 PCTAUS96/09455 (B) TYPE: nucleic acid (C) STR~Nn~nNF~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~u~ DESCRIPTION: SEQ ID NO:189:
' GCGGTAACCA GTACAAGGTG CTAAACGTAA TGGCGC 36 (2) INFORMATION FOR SEQ ID NO:190:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(Xi) ~U~N~: DESCRIPTION: SEQ ID NO:190:

(2) lN~O~JATION FOR SEQ ID NO:l91:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STR~N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l91:

(2) INFORMATION FOR SEQ ID NO:192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STR~N~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:

(2) INFORMATION FOR SEQ ID NO:193:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) STRPMn~n~.SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
(ix) FEATURE:
(D) OTHER INFORMATION: N at position 1 is an amino modifier C6 dT
(ix) FEATURE:
(D) OTHER INFORMATION: Nucleotide 51 is an inverted-orientation (3'3' linkage) phosphoramidite (xi) S~Qu~N-u~ DESCRIPTION: SEQ ID NO:193:

(2) INFORMATION FOR SEQ ID NO:194:

CA 0222327~ l997-l2-02 W 0 9~ 703 PCTA

(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STR~Nn~n~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:
TAGCCATTCA CCATGGCCCC TTCCTACGTA l~lL~rGCGG GTGGCTTA 48 (2) INFORMATION FOR SEQ ID NO:195:
U~N~ CHARACTERISTICS:
(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) sTRpNnRnNF~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~QU~:N-~ DESCRIPTION: SEQ ID NO:195:

(2) INFORMATION FOR SEQ ID NO:196:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: N at position 1 is an amimo modifier C6 dT
(ix) FEATURE:
(D) Oln~R INFORMATION: Nucleotide number 28 is an inverted-orientation (3'3' linkage) phosphoramidite (xi) S~YU~N-~ DESCRIPTION: SEQ ID NO:196:

(2) INFORMATION FOR SEQ ID NO:197:
(i) ~Q~:N-~ CHARACTERISTICS:
(A) LENGTH:40 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nNF~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:197:
TAATACGACT CACTATAGGG AGACAAGAAT A~ACGCTCAA 40 (2) INFORMATION FOR SEQ ID NO:198:
U~N-~ CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~.SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(Xi) ~U~N~ DESCRIPTION: SEQ ID NO:198:
GC~l~ll~lG AGCCTCCTGT CGAA 24 CA 0222327~ 1997-12-02 WO 9OE'~C17û3 PCI/US96/09455 (2) INFORMATION FOR SEQ ID NO:199:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STR~Nn~n~-~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) O~L~R INFORMATION: All U's are 2'-F uracil (Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:199:

(2) lN~oRrlATIoN FOR SEQ ID NO:200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (Xi) ~U~N~: DESCRIPTION: SEQ ID NO:200:
GGGAGACAAG AAUAAACGCU CAACGAG W C ACAUGGGAGC AAu~uCC~AA 50 UAAACAACAC GCKAKCGCAA A W CGACA&G AGGCUCACAA CAGGC 95 (2) INFORMATION FOR SEQ ID NO:201:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:
GGGAGACAAG AAUA~ACGCU CAACGACCAC AAUACAAACU CGUAUGGAAC 50 ACGCGAGCGA CAGUGACGCA uuuuCGACAG GAGGCUCACA ACAGGC 96 (2) INFORMATION FOR SEQ ID NO:202:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single - (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:

CA 0222327~ l997-l2-02 W O 9"~0703 PCTAUS~ 155 (D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~u~N~ DESCRIPTION: SEQ ID NO:202:
GGGAGACAAG AAUA~ACGCU CAACGUCAAG CCAGAAUCCG GAACACGCGA 50 GA~AACAAAU CAACGACCAA UCGA W CGAC AGGAGGCUCA CAAAGGC 97 (2) INFORMATION FOR SEQ ID NO:203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:203:

(2) INFORMATION FOR SEQ ID NO:204:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) sTRAMn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~U~N~ DESCRIPTION: SEQ ID NO:204:
GGGAGACAAG AAUA~ACGCU CAACGAACCA CGGGGAAAUC CACCAGUAAC 50 (2) INFORMATION FOR SEQ ID NO:205:
(i) ~u~N~ CH~RACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAlU~E:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2~-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:
GGGAGACAAG AAUA~ACGCU CAACGAGCAA AAGUACUCAC GGGACCAGGA 50 (2) INFORMATION FOR SEQ ID NO:206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ 1997-12-02 W O g~ 7~3 PCTrUS9~ g15' (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:206:

t2) INFORMATION FOR SEQ ID NO:207:
(i) S~:QU~N~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRAN~ l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil .(Xi) ~U~N-~ DESCRIPTION: SEQ ID NO:207:

(2) INFORMATION FOR SEQ ID NO:208:
(i) ~U~:N~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~U~N-~ DESCRIPTION: SEQ ID NO:208:

(2) INFORMATION FOR SEQ ID NO:209:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 101 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:

CA 0222327~ 1997-12-02 W 096l~0703 PCT/U~ 5 (2) INFORMATION FOR SEQ ID NO:210:
( i ) ~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:
(ix) FEATURE:
(D) OTHER lN~O~IATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:
GGGAGACAAG AAUA~ACGCU CAACAG W CA CUCAACCGGC ACCAGACUAC 50 (2) INFORMATION FOR SEQ ID NO:211:
(i) S~u~: CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~u~N~ DESCRIPTION: SEQ ID NO:211:
GGGAGACAAG AAUA~ACGCU CAACUGGCAA CGGGAUAACA ACA~AUGUCA 50 (2) INFORMATION FOR SEQ ID NO:212:
(i) S~u~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STR~N~ l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEAru~E:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:
GGGAGACAAG AAUA~ACGCU CAACGAUGAG CGUGACCGAA GCUAUAAUCA 50 (2) INFORMATION FOR SEQ ID NO:213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:

CA 0222327~ 1997-12-02 W O g6'4~703 PCTrUS96/09455 (D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:
GGGAGACAAG AAUAAACGCU CA~AGGAUCA CACA~ACAUC GGUCAAUAAA 50 (2) INFORMATION FOR SEQ ID NO:214:
U~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) O'l~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:

(2) INFORMATION FOR SEQ ID NO:215:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRAN~ S: single (D) TOPOLOGY:linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U~s are 2~-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:
GGGAGACAAG AAUA~ACGCU CAAGCGGUCA GAAACAAUAG CUGGAUACAU 50 (2) INFORMATION FOR SEQ ID NO:216:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:

(2) INFORMATION FOR SEQ ID NO:217:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 0222327~ l997-l2-02 W0 96/40703 PCT/U~5''~9155 ~ D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: A11 C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:217:

(2) INFORMATION FOR SEQ ID NO:218:
U~:N~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~u~N~ DESCRIPTION: SEQ ID NO:218:
GGGAGACAAG AAUA~ACGCU CAAUCAGCAG UAAGCGAUCC UAUA~AGAUC 50 (2) INFORMATION FOR SEQ ID NO:219:
( i ) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) m E: nucleir acid (C) STR~N~ :SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATuKE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:
GGGAGACAAG AAUA~ACGCU CAAAAAGACG UA W CGA W C GA~ACGAGAA 50 (2) INFORMATION FOR SEQ ID NO:220:
u~N~ CHARACTERISTICS:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) ~yu~N-~ DESCRIPTION: SEQ ID NO:220:
CUCAACGAAU CAGUA~ACAU AACACCAUGA A~CAUA~AUA GCACGCGAG 49 (2) INFORMATION FOR SEQ ID NO:221:

CA 0222327~ l997-l2-02 WO ~ '0703 PCT/U' 5 "05 !';~

( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:
CUCAACGAGU UCACAUGGGA GCAAUCUCCG AAUA~ACAAC ACGCGAG 47 (2) INFORMATION FOR SEQ ID NO:222:
(i) ~Yu~ CHARACTERISTICS:
(A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAruKE:
(D) Ol~R INFORMATION: All C's are 2~-F cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:222:
CUCAACGAAC CACGGGGA~A UCCACCAGUA ACACGCGAG 39 (2) INFORMATION FOR SEQ ID NO:223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~yu~N~ DESCRIPTION: SEQ ID NO:223:

(2) INFORMATION FOR SEQ ID NO:224:
(i) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:
CGCUCAACGA GCCAGGAACA UCGACGUCAG CA~ACGCGAG CG 42 CA 0222327~ 1997-12-02 W 096t40703 PCTtUS9C1'~5'~S

(2) INFORMATION FOR SEQ ID NO:225:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OT~R INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:225:
CUCAACGAGC CAGGACUACG AUCAGCA~AC GCGAG 35 (2) INFORMATION FOR SEQ ID NO:226:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTH~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:

(2) INFORMATION FOR SEQ ID NO:227:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid tC) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:

(2) INFORMATION FOR SEQ ID NO:228:
(i) S~u ~'N-~ CHARACTERISTICS:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEAT~KE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:228:

CA 0222327~ l997-l2-02 W O 3~ 3 PCT/U'~ S~'5 (2) INFORMATION FOR SEQ ID NO:229:
Qu~:N~ CHARACTERISTICS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~cs single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~u~N~ DESCRIPTION: SEQ ID NO:229:
GA~UCCGGAA CACGCGAGAA AACAAAUCAA CGACCAAUCG A W CG 45 (2) INFORMATION FOR SEQ ID NO:230:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) sT~Nn~n~R~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (ix) FEATURE:
(B) LOCATION: 7, 9, 14, 21 (D) Ol~R INFORMATION: G are 2'-O-methyl guanine (ix) FEAlu~E:
(B) LOCATION: 8, 15, 18, 22, 27, 31 (D) Ol~ER INFORMATION: A are 2'-O-methly ~n~ne (xi) S~u~N~: DESCRIPTION: SEQ ID NO:230:

~2) INFORMATION FOR SEQ ID NO:231:
u~N~ CHARACTERISTICS:
(A) LBNGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER lN~O~ ~TION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2~-F uracil (ix) FEATURE:
(B) LOCATION:7, 9, 13, 14, 21, 24, 28 (D) OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE:
(B) LOCATION:8, 15, 18, 22, 27, 30, 31 (D) OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:231:

(2) INFORMATION FOR SEQ ID NO:232:

CA 0222327~ 1997-12-02 WO ~ C703 PCTAU5~ 9'~5 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~CS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE:
(B) LOCATION: 7, 9, 14, 21, 36 (D) Ol~R INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE:
(B) LOCATION:8, 15, 18, 22, 27, 31, 37 (D) OTHER INFORMATION: A are 2'-O-methyl-A~nine (xi) S~:QU~N~ DESCRIPTION: SEQ ID NO:232:

(2) INFORMATION FOR SEQ ID NO:233:
(i) ~U~NC~: CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE:
(B) LOCATION: 7, 9, 13, 14, 21, 24, 28, 36 (D) OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE:
(B) LOCATION: 8, 15, 18, 22, 27, 30, 31, 37 (D) OTHER INFORMATION: A are 2'-O-methyl-A~ni (Xi) S~U~N~ DESCRIPTION: SEQ ID NO:233:

(2) INFORMATION FOR SEQ ID NO:234:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE:
(B) LOCATION:7, 9, 14 (D) OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEAlu~E:
(B) LOCATION:8, 15, 18, 27, 31 CA 0222327~ l997-l2-02 WO 9CJ~703 PCT/U'r 09~'~

(D) OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) S~yu N~: DESCRIPTION: SEQ ID NO:234:

(2) INFORMATION FOR SEQ ID NO:235:
~ (i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATuKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (ix) FEATURE:
(B) LOCATION: 7, 9, 13, 14, 24 (D) OTHER INFORMATION: G are 2'-O-methyl-guanine (ix) FEATURE:
(B) LOCATION: 8, 15, 18, 22, 27, 31 (D) OTHER INFORMATION: A are 2'-O-methyl-adenine (xi) S~Uk~: DESCRIPTION: SEQ ID NO:235:

(2) INFORMATION FOR SEQ ID NO:236:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~Qu~N~ DESCRIPTION: SEQ ID NO:236:

CGA~A WCG 59 (2) INFORMATION FOR SEQ ID NO:237:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAluKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~u~N~ DESCRIPTION: SEQ ID NO:237:

(2) INFORMATION FOR SEQ ID NO:238:
(i) S~u~ CHARACTERISTICS:

CA 0222327~ 1997-12-02 (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238:

(2) INFORMATION FOR SEQ ID NO:239:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:239:

(2) INFORMATION FOR SEQ ID NO:240:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid ~C) STRANn~N~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N-~ DESCRIPTION: SEQ ID NO:240:

(2) INFORMATION FOR SEQ ID NO:241:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241:

CA 0222327~ l997-l2-02 WO ~G/40703 PCT/US96/09455 (2) INFORMATION FOR SEQ ID NO:242:
( i ) S~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STR~N~ l)N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURB:
(D) Oln~:R INFORMATION: All C's are 2'-F cytosine (ix) FBATURB:
(D) OTHBR INFORMATION: All U's are 2'-F uracil (Xi) ~yu~N~ DESCRIPTION: SEQ ID NO:242:

(2) INFORMATION FOR SEQ ID NO:243:
( i ) ~U~N~' CHARACTERISTICS:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDBDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FBATURE:
(D) OTn~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURB:
(D) OTnBR INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:
ACGCUCAAAG GAUCACACAA ACAuCG~u~A AUAAAUAAGU A W GAUAGCG 50 (2) INFORMATION FOR SEQ ID NO:244:
U~N~ CHARACTERISTICS:
(A) LENGTn: 52 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi ) S~U~N~ DESCRIPTION: SEQ ID NO:244:
GCUCAAGCGG UCAGA~ACAA UAGCUGGAUA CAUACCGCGC AUCCGCUGGG 50 (2) INFORMATION FOR SEQ ID NO:245:
(i) SEQUBNCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
~ (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine CA 0222327~ 1997-12-02 WO 96/40703 PCT/U~i' ".~5 '~5 (iX) FEATURE
(D) OL~R INFORMATION: A11 UIS are 2'-F uracil (Xi) S~U~N~ DESCRIPTION SEQ ID NO:245:

ACCAUCUAGA GC W CGAACC AUGGUAUACA AGGGAACACA A~A W CGCGG 50 (2) INFORMATION FOR SEQ ID NO:246:
(i) ~Y~N~ CHARACTERISTICS
(A) LENGTH 96 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY: linear (ii) MO~ECULAR TYPE:RNA
(iX) FEATURE
(D) O~rn~R INFORMATION: A11 C~S are 2~-F cytosine (iX) FEATURE:

(D) Oln~R INFORMATION: A11 UIS are 2'-F uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO 246 (2) INFORMATION FOR SEQ ID NO:247:
(i) S~U~N-~ CHARACTERISTICS
(A) LENGTH 87 base pairs (B) TYPE nucleic acid (C) STRANIJ~ S single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION: A11 CIS are 2'-F cytosine (iX) FEATURE
(D) OTHER INFORMATION: A11 U~S are 2'-F uracil (Xi) S~U~N-.~ DESCRIPTION: SEQ ID NO 247 (2) INFORMATION FOR SEQ ID NO:248:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 97 base pairs (B) TYPE nucleic acid (C) STRAN~:~-N-~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION: A11 C~S are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION: A11 U~S are 2'-NH2 uracil (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:248:

GGGAGACAAG AAUAAACGCU CA~ NN~-N NN~NNNN~ ~NN~N~NN~ 50 NN~NN~N~ NN~N~NN NNNUUCGACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO:249:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 40 base pairs (s) TYPE: nucleic acid CA 0222327~ l997-l2-02 W O 9''~ 703 PCT/U~"0g1',5 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~QU~N~: DESCRIPTION: SEQ ID NO:249:

(2) INFORMATION FOR SEQ ID NO:250:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) ~,~yu~N~ DESCRIPTION: SEQ ID NO:250:
GC~ ; l L- r LG AGC~''LC~ L~:r L CGAA 24 (2) INFORMATION FOR SEQ ID NO:251:
(i) 'r~yU~:N~ CHARACTERISTICS:
(A) I,ENGTH: 97 base pairs (B) TYPE: nucleic acid (C) ST~'ANn~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251:
GGGAGACAAG AAUA~ACGCU CAAGCCCCAA ACGCAAGCGA GCAUCCGCAA 50 CAGGGAAGA~ GACAGACGAA UGA W CGACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO:252:
( i ) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRAN~ JN~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:252:

(2) INFORMATION FOR SEQ ID NO:253:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine CA 0222327~ l997-l2-02 W096t40703 PCT/U~5"~ S

(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253:
GGGAGACAAG A~AUA~ACNC UCAAGCCCCA AACGCAAGUG AGCAUCCGCA 50 (2) INFORMATION FOR SEQ ID NO:254:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~u~N~ DESCRIPTION: SEQ ID NO:254:

(2) INFORMATION FOR SEQ ID NO:255:
(i) S~u~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) sTRpNn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:255:

(2) INFORMATION FOR SEQ ID NO:256:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAlU~E:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:256:

(2) INFORMATION FOR SEQ ID NO:257:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid CA 0222327~ l997-l2-02 WO 96/40703 PCT/U~,~'03~.~5 ~ 189 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Qu~ DESCRIPTION: SEQ ID NO:257:
GGGAGACAAG AAUA~ACGCU CA~ACAGCUA CAAGUGGGAC AACAGGGUAC 50 AGCGGAGAGA AACAUCCA~A CAAGW CGAC AGGAGGCUCA CAACAGGC 98 (2) INFORMATION FOR SEQ ID NO:258:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STR~N~ )N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:258:
GGGAGACAAG AAUA~ACGCU CA~AUCAACU A~ACAACGCA GUCACGAGAA 50 CGACCGGKCU GACUCCGA~A G W CGACAGG AGGCUCACAA CAGGC 95 (2) INFORMATION FOR SEQ ID NO:259:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAlUKE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:259:
GGGAGACAAG AAUAAACGCU CA~ACGAGAG CACCAAGGCA ACAGAUGCAG 50 (2) INFORMATION FOR SEQ ID NO:260:
(i) S~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 98 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:260:
GGGAGACAAG AAUA~ACGCU CAAUAAGACA ACGAACAGAC AGAAGCGA~A 50 CA 0222327~ 1997-12-02 W O 9~''0703 PCT/U'3~'~5~'S

(2) INFORMATION FOR SEQ ID NO:261:
QU~N~: CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTXER INFORMATION: All C's are 2~-NHz cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All Uls are 2'-NH2 uracil (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:261:

(2) INFORMATION FOR SEQ ID NO:262:
(i) ~U~N~ CXARACTERISTICS:
(A) LENGTX: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-NX2 cytosine (ix) FEATURE:
(D) OTXER INFORMATION: All Uls are 2'-NH2 uracil (Xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:262:
GGGAGACAAG AAUA~ACGCU CAAACA~AAU uwu w u~GGC CCCGCAACMG 50 (2) INFORMATION FOR SEQ ID NO:263:
(i) S~QU~N~ CXARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NX2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All Uls are 2'-NX2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:263:

(2) INFORMATION FOR SEQ ID NO:264:
( i ) S~ U ~:N~ CXARACTERISTICS:
(A) LENGTX: 89 base pairs (B) TYPE: nucleic acid (C) STRANDEDNES S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEALuKE:

CA 0222327~ 1997-12-02 WO 9~1C703 PCT/US96 (D) OTHER INFORMATION: All C ' s are 2 '-NH2 cytosine ( ix ) FEATURE:
(D) OTHER INFORMATION: All U' s are 2 ' -NH2 uraeil (xi) ~ U~;N~:~; DESCRIPTION: SEQ ID NO:264:
- GGGAGACAAG AAUA~ACGW CA~ACACA~A UCGGGCAGGG AWGGGWGG 50 - GCACGGCAGG GCGC~:uuC~A CAGGAGGWC ACAACAGGC 89 (2) INFORMATION FOR SEQ ID NO:265:
;UU~;N~:~; CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nueleic aeid (C) STRANV~;VN~;ss: single (D) TOPOLOGY: linear ( ii ) MOLECIJLAR TYPE: RNA
( ix ) FEATURE:
(D) OTHER INFORMATION: All C ' s are 2 ' -NH2 cytosine ( ix ) FEATURE:
(D) OTHER INFORMATION: All U' s are 2 ' -NH2 uraeil (xi) ~;UU~;NC ~; DESCRIPTION: SEQ ID NO:265:
GGGAGACAAG AAUA~ACGW CAAGUGGGW CGGGCCGGAU GUWACGGGU 50 (2) INFORMATION FOR SEQ ID NO:266:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLEWLAR TYPE:
( ix ) FEATURE: RNA
(D) OTHER INFORMATION: All C ' s are 2 ' -NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U' s are 2 '-NH2 uracil (xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID NO:266:
GGGAGACAAG AAUA~ACGCU CAAGAUCAGC GGAAWAAGA AAUGGAAGGC 50 (2) INFORMATION FOR SEQ ID NO:267:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( i i ) MOLECULAR TYPE: RNA
( ix ) FEAl l~E:
(D) OTHER INFORMATION: All C ' s are 2 ' -NH2 cytosine ( ix) FEATU~E:
(D) OTHER INFORMATION: All U' s are 2 ' -NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:267:
GGGAGACAAG AAUA~ACGCU CAAUAACAAA GCAGCAAAGU ACCAGAGGAG 50 (2) INFORMATION FOR SEQ ID NO:268:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 95 base pairs CA 0222327~ l997-l2-02 WO 9"'~703 PCT/U' 3GI'~S ~5.S

(B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAT~E:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) S~U~N~ DESCRIPTION: SEQ ID NO:268:
GGGAGACAGA AUA~ACGCUC A~AGACCAAG GGACAGCAGC GGGGA~AAAC 50 (2) INFORMATION FOR SEQ ID NO:269:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 93 base pairs (B) TYPE: nucleic acid (C) STR~Nn~n-N~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:269:
GGGAGACAAG AAUAAACGCU CA~AGUCGGG GAUAGA~ACA CACUAAGAAG 50 (2) INFORMATION FOR SEQ ID NO:270:
(i) S~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) sTRpNn~n-N-~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:270:
GGGAGACAAG AAUA~ACGCU CAAGAGUAUC ACACA~ACCG GCACGGACUA 50 (2) INFORMATION FOR SEQ ID NO:271:
(i) ~U~N~ CHARACTERISTICS:
(A~ LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) ~U~N-~: DESCRIPTION: SEQ ID NO:271:
GGGAGACAAG AAUAAACNCU CAACGA~AUA GAAGGAACAG AAGAAUGGBG 50 CA 0222327~ 1997-12-02 W O 96/40703 PC~rAUS96/09455 AWGNGGGA~A UGGCAACGAA W CGACAGGN GGCUCACAAC AGGC 94 (2) INFORMATION FOR SEQ ID NO:272:
(i) S~yU~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~cs single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:272:
GGGAGACAAG AAUAAACGCU CA~ACGAGAC CCUGGAUACG AGGCUGAGGG 50 A~AGGGAGMM MRRAMCUARR CKC W CGACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO:273:
yU~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAlu~E:
(D) Ol~ER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~YU~N-~ DESCRIPTION: SEQ ID NO:273:
GGGAGACAAG AAUA~ACGCU CAAGAAGGAU AC WAGGACU ACGUGGGAUG 50 GGAUGA~AUG GGAGAACGGG AG W CGACAG GAGGCUCACA ACAGGC 96 (2) INFORMATION FOR SEQ ID NO:274:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (Xi) S~:QU~N~ DESCRIPTION: SEQ ID NO:274:
GGGAGACAAG AAUA~ACGCU CAAAACGCAC AAAGUAAGGG ACGGGAUGGA 50 (2) INFORMATION FOR SEQ ID NO:275:
(i) s~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid ~ (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:

CA 0222327~ 1997-12-02 W O 96/40703 PCTrUS9''~ ~ee (D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:275:

(2) INFORMATION FOR SEQ ID NO:276:
( i ) S~QU~N~'~: CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STR~N~ )N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:276:
GGGAGACAAG AAUA~ACGCU CAAUGAUAUA CACGUAAGCA CUGAACCAGG 50 ~2) INFORMATION FOR SEQ ID NO:277:
u~ CHARACTERISTICS:
(A) LENGTH: 94 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) O'l~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:277:
GGGAGACAAG AAUA~ACGCU CAAGAUCAUA ACGAGAGGAG AGGGAGAACU 50 (2) INFORMATION FOR SEQ ID NO:278:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STRA~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~U~N-~ DESCRIPTION: SEQ ID NO:278:
GGGAGACAAG AAUAAACGCU CAAUCAAGUA AGGAGGAAGG ~uC~uGACAG 50 (2) INFORMATION FOR SEQ ID NO:279:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 93 base pairs CA 0222327~ 1997-12-02 W O 9G'~703 PCT/U~,.'J91~5 (B) TYPE nucleic acid (C) STRANV~VN~SS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE:RNA
(iX) FEATURE
(D) OTHER 1N~-O~IATION: A11 C~S are 21-NH2 cytosine (iX) FEATURE:
(D) OTHER INFORMATION A11 UIS are 21-NH2 uracil (Xi) ~QU~N~ DESCRIPTION SEQ ID NO:279:

(2) INFORMATION FOR SEQ ID NO 2 80 QU~N~ CHARACTERISTICS
(A) LENGTH 97 base pairs (B) TYPE nucleic acid (C) STRANn~nN~SS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 21-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 UIS are 21-NH2 uracil (Xi) S~U~N~ DESCRIPTION SEQ ID NO:280:
GGGAGACAAG AAUAAACGCU CAACCAACGC GCACCCCGCA GCA~ACGAAA 50 (2) INFORMATION FOR SEQ ID NO:281:
(i) ~QU~N~ CHARACTERISTICS
(A) LENGTH: 97 base pairs (B) TYPE nucleic acid (C) STRANn~nN~S single (D) TOPOLOGY 1 inear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2'-NH2 cytosine (iX) FEATURE
(D) OTHER INFORMATION A11 UIS are 21-NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:281:
GGGAGACAAG AAUA~ACKCU CAACAAACAA UAUCGGCGCA GGAAAACGUA 50 GAAACGA~AM GGAGCUGCGY GGA W CGACA GGAGGCUCAC AACAGGC 97 (2~ INFORMATION FOR SEQ ID NO:282:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 93 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE RNA
(iX) FEATURE
(D) OTHER INFORMATION A11 CIS are 2' -NH2 cytosine . (iX) FEATURE
(D) OTHER INFORMATION A11 UIS are 2' -NH2 uracil (Xi) SEQUENCE DESCRIPTION SEQ ID NO:282 CA 0222327~ 1997-12-02 WO 9~'1C /u3 PCT/US9''~S ~5' (2) INFORMATION FOR SEQ ID NO:283:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs (B) TYPE: nucleic acid (C) STR~NnRnN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~:QU~N-~: DESCRIPTION: SEQ ID NO:283:

(2) INFORMATION FOR SEQ ID NO:284:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) Or~R INFORMATION: All C's are 2~-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:284:

(2) INFORMATION FOR SEQ ID NO:285:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~QU~N~ DESCRIPTION: SEQ ID NO:285:

(2) INFORMATION FOR SEQ ID NO:286:
(i) ~QU~N-~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:

CA 0222327~ l997-l2-02 WO g~'~t703 PCr/U~CI'O9'-~

(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) ~Q~N~ DESCRIPTION: SEQ ID NO:286:

~ AAGGGGCAGC GCGAAGAUCA CAAW CGACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO:287:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NHz uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:287:
GGGAGACAAG AAUAAACGCU CAAGGGA~AC GGAAAGGGAC AAGGCGAACA 50 (2) INFORMATION FOR SEQ ID NO:288:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRPNn~nNRqS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~u~N~ DESCRIPTION: SEQ ID NO:288:

(2) INFORMATION FOR SEQ ID NO:289:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 95 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEAL~E:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEAluKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 uracil (xi) S~Qu~N-~ DESCRIPTION: SEQ ID NO:289:
GGGAGACAAG AAUA~ACGCU CAAGUACRCA GUGAGCAGAA GCAGAGAGAC 50 (2) INFORMATION FOR SEQ ID NO:290:
Qu~N~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs CA 0222327~ 1997-12-02 W O g6'1D703 PCTAUS96/09455 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) Olh~R INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-NH2 uracil (xi) ~U~N~: DESCRIPTION: SEQ ID NO:290:

ACCAGGCUGN BCNGCACCAS AC~uu~ACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO:291:
U~N~ CHARACTERISTICS:
(A) LENGTH: 11 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE:RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-NH2 cytosine (ix) FEATURE:
(D) OTHER lN~O~IATION: All U's are 2'-NH2 uracil (xi) S~u~ DESCRIPTION: SEQ ID NO:291:

(2) INFORMATION FOR SEQ ID NO:292:
(i) S~yu~ CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlUKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:292:
GGGAGGACGA UGCGGNNNNN NI~NNNNNNN~ ~N~NNNNN~I NNNNNN~NNN 50 (2) INFORMATION FOR SEQ ID NO:293:
U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:293:

CA 0222327~ l997-l2-02 WO 9~ 703 PCT/U~ C5 GGGAGGACGA UGCGGGCA~A W GCAUGCGU WW CGAGUGC W GCUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:294:
( i ) ~OU~N~ CHARACTERISTICS:
- (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTn~R INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~yU~N~ DESCRIPTION: SEQ ID NO:294:
GGGAGGACGA UGCGGUGCUU A~ACAACGCG UGAAUCGAGU UCAUCCACUC 50 ~u~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:295:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Orn~R INFORMATION: All C~s are 2~-F cytosine (ix) FEATURE:
(D) OLn~R INFORMATION: All U's are 2'-F uracil (Xi) S~UU~N-~ DESCRIPTION: SEQ ID NO:295:

(2) INFORMATION FOR SEQ ID NO:296:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:296:

(2) INFORMATION FOR SEQ ID NO:297:
( i ) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs i (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECUhAR TYPE: RNA

CA 0222327~ l997-l2-02 W O ~6,~703 PCT/U',.'~9155 tix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:297:
GGGAGGACGA UGCGGUACCU CAPA W GCGU ~uUuU~AAGC AGUAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:298:
( i ) ~UU~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) sTRpNnRnNR~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OL~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) S~U~N~: DESCRIPTION: SEQ ID NO:298:
GGGAGGACGA UGCGGACCCU CAPAUAACGU ~u~uuu~AAG W GGUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:299:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRPN~RnN~.SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:299:

(2) INFORMATION FOR SEQ ID NO:300:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:300:
GGGAAGACGA UGCGGCGCUC A~AUAAUGCG W AAUCGAAU UCGCCCAGAC 50 (2) INFORMATION FOR SEQ ID NO:301:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ l997-l2-02 WO g~ 703 PCT/US96/09455 (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
- (ix) FEATURE:
(D) Ol~:R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) S~yU~N~ DESCRIPTION: SEQ ID NO:301:
GGGAGGACGA UGCGGCAAAC AAGCUCAAAU GAC~u~uuuu UCAAGUCC W 50 ~uu~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:302:
( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRP~ S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATuKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:302:

(2) INFORMATION FOR SEQ ID NO:303:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAluKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEAlU~E:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:303:
GGGAGGACGA UGCGGAGACU CAAAUGGUGU ~Uuuu~AAGC CUCUCCCAGU 50 (2) INFORMATION FOR SEQ ID NO:304:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 63 base pairs . (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~yu~N~ DESCRIPTION: SEQ ID NO:304:

CA 0222327~ l997-l2-02 W O g6"C703 PCTAUS96/09455 GGGAGGACGA UGCGGUGCUC A~AUGAUGCG uuu~u~AAU C QCC QGAC 50 (2) INFORMATION FOR SEQ ID NO:305:
(i) S~:Q~ ~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yU~N~ DESCRIPTION: SEQ ID NO:305:
GGGAGGACGA UGCGGCCAUC G~u~uu~GGC AACGCGU W U CGAG W ACCU 50 (2) INFORMATION FOR SEQ ID NO:306:
u~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OLn~R INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:306:
GGGAGGACGA UGCGGC QUC G~u~uu~GGC AACGCGU W U CGAGUUACCU 50 (2) INFORMATION FOR SEQ ID NO:307:
(i) s~yu~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) s~yu~N~ DESCRIPTION: SEQ ID NO:307:
GGGAGGACGA UGCGGGACCC W AGGCAACG u~uuuu~AAG W GGUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:308:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

CA 0222327~ l997-l2-02 W O g~ 703 PCT/U'36~'05'~

(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~QU~N~ DESCRIPTION: SEQ ID NO:308:

u~u~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:309:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) ST~NnR~N~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:309:
GGGAGGACGA UGCGGAGUCU UAGGCAGCGC ~uuuuCGAGC UACUCCAUCG 50 (2) INFORMATION FOR SEQ ID NO:310:
yu~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) S~YU~N~ DESCRIPTION: SEQ ID NO:310:

(2) INFORMATION FOR SEQ ID NO:311:
( i ) S~U~N~'~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:311:
GGGAGGACGA UGGGGAGUCU UAGGCAGCGC ~uuuuCGAGC UACUCCAUCG 50 (2) INFORMATION FOR SEQ ID NO:312:
(i) SEQUENCE CHARACTERISTICS:

WO ~/4C703 PCT/U' ,~ 5 (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~N~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Oln~R INFORMATION: All U's are 2'-F uracil (xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:312:

(2) INFORMATION FOR SEQ ID NO:313:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~u~ DESCRIPTION: SEQ ID NO:313:
GGGAGGACGA UGCGGCAAUG u~uuAGGC CACGCG W AA UCGAGCGUGA 50 (2) INFORMATION FOR SEQ ID NO:314:
(i) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:314:

(2) INFORMATION FOR SEQ ID NO:315:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~QD~N~ DESCRIPTION: SEQ ID NO:315:

CA 0222327~ l997-l2-02 W O 9~'~C703 PCTAJ~9'~r1~' GGGAGGACGA UGCGGGAUGC UUAGGCGCCG U~UUUU~AAG GCCAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:316:
U~ CHARACTERISTICS:
(A) LBNGTH: 72 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRAN~N~SS: Sing1e (D) TOPOLOGY: 1inear (ii) MOLECULAR TYPE: RNA
(iX) FEATURE:
(D) OTHER INFORMATION: A11 CIS are 2l-F CYtOSine (iX) FEATURE:
(D) O1~R INFORMATION: A11 U~S are 2l-F UraCi1 (Xi) S~QU~N-~ DESCRIPTION: SEQ ID NO:316:
GGGAGGACGA UGCGGUAA W GUCUUAGGCG CC~UW UAUC AAGGCACAAU 50 (2) INFORMATION FOR SEQ ID NO:317:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRA~ S Sing1e (D) TOPOLOGY: 1inear (ii) MOLBCULAR TYPE: RNA
(iX) FEATURE:
(D) OTHER INFORMATION: A11 CIS are 2l-F CYtOSine (iX) FEATURB:
~D) OTHER INFORMATION: A11 UIS are 2l-F UraCi1 (Xi) S~U~N~ DESCRIPTION: SEQ ID NO:317:
GGGAAGACGA UGCGGCUACU A~U~U~UUAG GCGGAGUG W UAUCAAUCCA 50 (2) INFORMATION FOR SEQ ID NO:318:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRAN~N~:SS: Sing1e (D) TOPOLOGY: 1inear (ii) MOLECULAR TYPE: RNA
(iX) FBATURE:
(D) OTHER INFORMATION: A11 CIS are 2~-F CYtOSine (iX) FEATURB:
(D) OTHER INFORMATION: A11 U~S are 2'-F UraCi1 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:318:

(2) INFORMATION FOR SEQ ID NO:319:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 70 baSe PairS
(B) TYPE: nUC1eiC aCid (C) STRANDEDNBS S: s ing1e (D) TOPOLOGY: 1inear (ii) MOLECULAR TYPE: RNA

CA 0222327~ l997-l2-02 W O 9~'~t703 PCT/U~ 5 (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~:~U~N~ DESCRIPTION: SEQ ID NO:319:
GGGAGGACGA UGCGGUGGUG u~u~uuu~GC ACCGCGUA W W CGAGGUAC 50 (2) INFORMATION FOR SEQ ID NO:320:
(i) S~:Qu~N~: CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) ST~N~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) O'l~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:320:
GGGAGGACGA UGCG~u~u~ u~u~uuu~GC ACCGCGUA W CUCGAGGUAC 50 ~2) INFORMATION FOR SEQ ID NO:321:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:321:

(2) INFORMATION FOR SEQ ID NO:322:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) sTRANnRn-NR~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xl) ~QU~N~ DESCRIPTION: SEQ ID NO:322:

(2) INFORMATION FOR SEQ ID NO:323:
( i ) ~QU~N~ CHARACTERISTICS:

CA 0222327~ l997-l2-02 W O 96/40703 PCT/U~ S'~' ~A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRaNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Orn~R INFORMATION: All U~s are 2'-F uracil (xi) S~yU~N~ DESCRIPTION: SEQ ID NO:323:

(2) INFORMATION FOR SEQ ID NO:324:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTn~R INFORMATION: All U's are 2'-F uracil (Xi) S~YU~N~ DESCRIPTION: SEQ ID NO:324:

(2) INFORMATION FOR SEQ ID NO:325:
( i ) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:325:

uu~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:326:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEAlu~E:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:326:

CA 0222327~ l997-l2-02 WO 9C"~03 PCT/U~,C~ ?~

(2) INFORMATION FOR SEQ ID NO:327:
(i) S~Qu~:N~: CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) sTRANn~n-N~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~U~N~ DESCRIPTION: SEQ ID NO:327:

(2) INFORMATION FOR SEQ ID NO:328:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) STRAN~N~:SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OT~:R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~yu~N~ DESCRIPTION: SEQ ID NO:328:

(2) INFORMATION FOR SEQ ID NO:329:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:329:

C~u~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:330:
(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

CA 0222327~ l997-l2-02 WO 9~ 03 PCT/U~ 9 ~15S

(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Oln R INFORMATION: All U's are 2'-F uracil (xi) S~yu~ DESCRIPTION: SEQ ID NO:330:

(2) INFORMATION FOR SEQ ID NO:331:
( i ) S~yu~N~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Oln~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Oln~R INFORMATION: All U's are 2'-F uracil (xi) ~yu~ DESCRIPTION: SEQ ID NO:331:
GGGAGGACGA UGCGGUGCUA W C W AAGCG GC~u~uuuuu CAAGCCAAUA 50 (2) INFORMATION FOR SEQ ID NO:332:
(i) ~Qu~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:332:
GGGAGGACGA UGCG~u~uuA AGCGGCGCGA W W CGAGCC ACCGCAUCCU 50 (2) INFORMATION FOR SEQ ID NO:333:
(i) ~yu~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRA~ :cs: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:333:
GGGAGGACGA UGCGGCCUCU UAAGCGUCGU ~uuuuu~AAG CUGGUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:334:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ l997-l2-02 W O 9t'~703 PCTAUS96/09455 (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: A11 U's are 2'-F uracil (Xi) S~YU~N~ DESCRIPTION: SEQ ID NO:334:

AUGGUCAGAC GA~uCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:335:
( i ) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRAN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER lN~O~ ~TION: All U~s are 2~-F uracil (Xi) ~:yU~N~ DESCRIPTION: SEQ ID NO:335:
GGGAGGACGA UGCGGUGCUA W C WAAGCG GCGUGUAAAU CA~GCUAGAU 50 (2) INFORMATION FOR SEQ ID NO:336:
( i ) ~yU~N~'~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(lx) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~YU~N.~ DESCRIPTION: SEQ ID NO:336:
GGGAGGACGA UGCGGAACGA CUCUUAAGCU GUGC~uuuuC GAACAAGUCG 50 (2) INFORMATION FOR SEQ ID NO:337:
U~NU~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: A11 U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:337:

CA 0222327~ l997-l2-02 W O 9~"G703 PCT/U~ 91'~

GACUCGCCCG A 6l (2) INFORMATION FOR SEQ ID NO:338:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLE W LAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~Qu N~'~ DESCRIPTION: SEQ ID NO:338:
GGGAGGACGA UGCGGAGUCW ~u~u~ACCA KC~u~uKuuA AUCAAG WAN 50 UGC W CAGAC GA W CGCCCG A 7l (2) INFORMATION FOR SEQ ID NO:339:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAMn~n~S: single (D) TOPOLOGY: linear (ii) MOLE W LAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~UU~N~ DESCRIPTION: SEQ ID NO:339:
GGGAGGACGA UGCGGU WAC G~U~u~u~uG GCGGUGCGUA AAUCKAACCA 50 GAUCGCAGAC GA W CGCCCG A 7l (2) INFORMATION FOR SEQ ID NO:340:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLE W LAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEAlu~E:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~U~N-~ DESCRIPTION: SEQ ID NO:340:

(2) INFORMATION FOR SEQ ID NO:341:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLE W LAR TYPE: RNA

CA 0222327~ l997-l2-02 W O 9~ 703 PCT/U~ 91 (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~: DESCRIPTION: SEQ ID NO:341:
GGGAGGACGA UGCGGAUCGC AAUMUGUWGC ~uu~u~KAAA CAGCCUCAGA 50 (2) INFORMATION FOR SEQ ID NO:342:
(i) ~yU~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRpNn~nN~s: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Oln~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~.yU~N~ DESCRIPTION: SEQ ID NO:342:
GGGAGGACGA UGCGGUGG W CUAGGCACGU ~uuuu~AAGU GUAAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:343:
(i) S~:QU~N~ CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRPNn~nN~-~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:343:
GGGAGGACGA UGCGGAAACA U~U~UUUUCG AAUGUGCUCU CCUCCCCA~A 50 (2) INFORMATION FOR SEQ ID NO:344:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEAlu~E:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:344:
GGGAGGACGA UGCGGAAGGC C~u~uuAAUC AAGGCUGCAA UAAAUCAUCC 50 (2) INFORMATION FOR SEQ ID NO:345:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ l997-l2-02 W O 9~/~a703 PCT/U~ 5''' (A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Ol~R INFORMATION: All U's are 2'-F uracil (Xi) S~yu N~ DESCRIPTION: SEQ ID NO:345:
GGGAGGACGA UGCGGAGGAU C~U~UU~AUC AAGA W GCUC ~UU-UUUACU 50 (2) INFORMATION FOR SEQ ID NO:346:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:346:
GGGAGGACGA UGCGGUCA~A GUGAAGAAUG GACAGCG W U UCGAG W GCU 50 (2) INFORMATION FOR SEQ ID NO:347:
( i ) ~yU~N~'~ CH~RACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~:QU~:N~ DESCRIPTION: SEQ ID NO:347:

(2) INFORMATION FOR SEQ ID NO:348:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) O'l~R INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:348:

CA 0222327~ 1997-12-02 WO 9CJ4û703 PCT/U~ s ~rr (2) INFORMATION FOR SEQ ID NO:349:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~N~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) Or~R INFORMATION: All U's are 2'-F uracil (xi) S~YU~N~ DESCRIPTION: SEQ ID NO:349:
GGGAGGACGA UGCGGAUCGA WW CAUGCGU uuuuC~AGUG ACGAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:350:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~NnRnN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:350:
GGGAGGACGA UGCGGAGACC CUAAGMGSGU K~uuuu~AAS ~U~U~'W~AC 50 (2) INFORMATION FOR SEQ ID NO:351:
(i) ~Qu~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~Q~N~ DESCRIPTION: SEQ ID NO:351:

(2) INFORMATION FOR SEQ ID NO:352:
(i) S~:QU~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

CA 0222327~ l997-l2-02 WO ~/4~703 PCT/U'~ 5.

(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~Qu~ DESCRIPTION: SEQ ID NO:352:
GGGAGGACGA UGCGG WAGG UCAAUGAUCU UA~uuuu~GA uuC~u~AGAC 50 (2) INFORMATION FOR SEQ ID NO:353:
u~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:353:
GGGAGGACGA UGCGGACGUG UGUAUCRARU uuuCCG~u~u W GUGCAGAC 50 (2) INFORMATION FOR SEQ ID NO:354:
(i) S~UU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAN~ l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:354:
GGGAGGACGA UGCGGACAGG ~uu~uuAGGC GGA~u~uu~A UCAAUCC~AC 50 (2) INFORMATION FOR SEQ ID NO:355:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:355:

(2) INFORMATION FOR SEQ ID NO:356:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ 1997-12-02 W O 9.'_0703 PCT/U~r~'0r~r5 (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid .
(C) STR~NnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~U~N~ DESCRIPTION: SEQ ID NO:356:
GGGAGGACGA UGCGGAUAYU CAG~uY~u~u Kuuuu~AUC W CCCCAGAC 50 (2) INFORMATION FOR SEQ ID NO:357:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~N~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:357:
GGGAGGACGA UGCGGCACAC ~u~uuuu~AA GUGUGCUCCU GGGAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:358:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~NnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:358:
GGGAGGACGA UGCGGCAAUG u~uuuuu~AA A W GCU W CU CCC W CAGAC 50 (2) INFORMATION FOR SEQ ID NO:359:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:359:

CA 0222327~ l997-l2-02 W O 96/40703 PCT/U'~'1~5~5' GGGAGGACGA UGCGGAUACU ACCGUGCGAA CACUAAGUCC C~U~U~UC~A 50 ~uC~u~AGAC GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:360:
U~N~ CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:360:
GGGAGGACGA UGCGGAUACU AUGUGCG W C ACUAAGUCCC ~U~UCCC~u 50 (2) lN~O~IATION FOR SEQ ID NO:361:
( i ) S~QU~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid ( C ) ST~Z~N I )~:1 IN l- ~S single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:361:

(2) INFORMATION FOR SEQ ID NO:362:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATuKE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:362:
GGGAGGACGA UGCGGUUACU AUGUACAU~U ACUAAGACCC AACGUCAGAC 50 (2) INFORMATION FOR SEQ ID NO:363:
( i ) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRPNn~N~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

CA 0222327~ 1997-12-02 W O 96/~ 03 PCT/U~3~ 5 (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATuKE:
(D) OTHER INFORMATION: A11 U's are 2'-F uracil (Xi) S~U~N'~ DESCRIPTION: SEQ ID NO:363:
GGGAGGACGA UGC~w uw~u AUGUw-CGCCU UACUAAGUAC CCGuCGACUG 50 (2) INFORMATION FOR SEQ ID NO:364:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil Ixi) ~U~N~'~ DESCRIPTION: SEQ ID NO:364:
GGGAAGACGA UGCGGUG W G AUCAAUGAAU ~UC~'U~UCC UACCCCAGAC 50 (2) INFORMATION FOR SEQ ID NO:365:
u~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) sTR~Nn~nN~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2~-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~QU~N-'~ DESCRIPTION: SEQ ID NO:365:

(2) INFORMATION FOR SEQ ID NO:366:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~U~N~'~ DESCRIPTION: SEQ ID NO:366:

(2) INFORMATION FOR SEQ ID NO:367:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ l997-l2-02 W O 9r'~G703 PCT/U'9~'v~

(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRPNnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
~ (ix) FEATURE:
(D) Orn~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~:N-~ DESCRIPTION: SEQ ID NO:367:

(2) INFORMATION FOR SEQ ID NO:368:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) STRPNnRnNR~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~ DESCRIPTION: SEQ ID NO:368:
GGGAGGACGA UGCGGAAUGR CCCG W ACCA WCAAUGCGCC U~uu~MCCC 50 (2) INFORMATION FOR SEQ ID NO:369:
(i) S~Q~N~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:369:
GGGAGGACGA UGCGGAAUYU C~u~YuACGC ~UYYY~UAUC CAAUCUACCC 50 (2) INFORMATION FOR SEQ ID NO:370:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:370:

CA 0222327~ 1997-12-02 WO ~ C703 PCT/US~'05 '~

(2) INFORMATION FOR SEQ ID NO:371:
(i) S~u~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STR~N~ l-N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: A11 C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) S~UU~N~ DESCRIPTION: SEQ ID NO:371:

(2) INFORMATION FOR SEQ ID NO:372:
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANn~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:372:
GGGAGGACGA UGCGGCACAA u~uuCGGCAG CGUGCAAGAU CAAGCUA W G 50 (2) INFORMATION FOR SEQ ID NO:373:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~u~ DESCRIPTION: SEQ ID NO:373:

(2) INFORMATION FOR SEQ ID NO:374:
:QukN~ CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

CA 0222327~ l997-l2-02 W O 9~'~C~03 PCT/U~''05~5.

(ix) FEALuKE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~QU~N~'~ DESCRIPTION: SEQ ID NO:374:
'~ GGGAGGACGA UGCGGUCA W CUCUAAAAAA GUAuuCC~uA CCUCCACAGA 50 (2) INFORMATION FOR SEQ ID NO:375:
(i) S~u~:N~h CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRPN~ N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OL~R INFORMATION: All U's are 2'-F uracil (xi) ~QU~N~'~ DESCRIPTION: SEQ ID NO:375:
GGGAGGACGA UGCGGGUGAU CU W UAUGCU C~u~uu~uuu C~u~u~AGAC 50 (2) INFORMATION FOR SEQ ID NO:376:
(i) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRPNI~:l)N~:~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) Ol~R INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:376:

(2) INFORMATION FOR SEQ ID NO:377:
(i) S~:Qu~N~: CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) sTRaNl~l)N~s single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(lx) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEA'l'UKE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~U~N~ DESCRIPTION: SEQ ID NO:377:

(2) INFORMATION FOR SEQ ID NO:378:
(i) SEQUENCE CHARACTERISTICS:

CA 0222327~ l997-l2-02 WO 9C,'~703 PCTrUSg6'0~4'C

(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:378:
GGGAGGACGA UGCGGCUCUC AUAUK~w~uK uuYuu~M W C SRGGCUCAAA 50 (2) INFORMATION FOR SEQ ID NO:379:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRPMn~nN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (Xi) ~h~U~ DESCRIPTION: SEQ ID NO:379:
GGGAGGACGA UGCGGCUUGU UAG WAAACU CGA~u~uC~A CCC~u~AGAC 50 (2) INFORMATION FOR SEQ ID NO:380:
(i) ~yu~ CHARACTERISTICS:
(A) LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANv~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N-~ DESCRIPTION: SEQ ID NO:380:
GGGAGGACGA UGCG~u~u~u WCUVACVUGU R W CACA WW UCGCYUCAAA 50 (2) INFORMATION FOR SEQ ID NO:381:
U~N~ CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:381:

CA 0222327~ l997-l2-02 W O 96/40703 PCT/U'3G/~5~'5 GGGAGGACGA UGCGG WRAC AAUGRSSCUC RC~uuCC~wG ~uC~u~AGAC 50 (2) INFORMATION FOR SEQ ID NO 382 (i) ~U~N-~ CHARACTERISTICS
- (A~ LENGTH 71 base pairs (B) TYPE nucleic acid (C) STRA~N~SS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(ix) FEATURE
(D) OTHER INFORMATION All C's are 2'-F cytosine (ix) FEATURE
(D) O'l~R INFORMATION All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION SEQ ID NO 382 AGGAGGACGA UGCGG W AUC UGAARCWUGC GUA~MCUARU GUSA~ASUGC 50 (2) INFORMATION FOR SEQ ID NO 383 (i) ~u~ CHARACTERISTICS
(A) LENGTH 61 base pairs (B) TYPE nucleic acid (C) STR~Mn~nN~S single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(ix) FEATURE
(D) OTHER INFORMATION All C's are 2'-F cytosine (ix) FEATURE
(D) OTHER INFORMATION All U's are 2'-F uracil (xi) ~yU~N~ DESCRIPTION SEQ ID NO 383 AGGAAGACGA UGCG~uuC~A W UA WW GUG UCAUU~uu~u UCCAUCAGAC 50 (2) INFORMATION FOR SEQ ID NO 384 (i) ~U~N~ CH~RACTERISTICS
(A) LENGTH 35 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(ix) FEATURE
(D) OTHER INFORMATION All C's are 2'-F cytosine (ix) FEATURE
(D) OTHER INFORMATION All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION SEQ ID NO 384 (2) INFORMATION FOR SEQ ID NO 385 OU~N-~ CHARACTERISTICS
(A) LENGTH 16 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULAR TYPE RNA
(ix) FEATURE

CA 0222327~ l997-l2-02 W O ~ D703 PCT/U55~ ~5 (D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~QD~:N~ DESCRIPTION: SEQ ID NO:385:
UGC~u~uuuu CAAGCA 16 (2) INFORMATION FOR SEQ ID NO:386:
~U~N~ CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRAN~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) S~U~N~ DESCRIPTION: SEQ ID NO:386:
CUCAAA W GC ~u~uuuu~AA GCA 23 (2) INFORMATION FOR SEQ ID NO:387:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~.cs: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) ~QU~N-~ DESCRIPTION: SEQ ID NO:387:

(2) INFORMATION FOR SEQ ID NO:388:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:388:

(2) INFORMATION FOR SEQ ID NO:389:
(i) ~U~N-~ CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s single (D) TOPOLOGY: linear ~ii) MOLECULAR TYPE: RNA

W O 9~ 703 PCT/U~ 9~55 (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-F uracil (xi) SEQUENCE DESCRIPTION: SEQ ID NO:389:
GGGAGGACGA UGcG~-~N~NN~ TNNN~NNNN ~NN~ ~3~NI~IN~-NN~N 50 NNNNN~AGAc GACUCGCCCG A 71 (2) INFORMATION FOR SEQ ID NO:390:
(i) S~YU~N-~ CHARACTERISTICS:
(A) LENGTH: 97 base pairs (B) TYPE: nucleic acid (C) STRPNI~:I)N~ S single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F cytosine (ix) FEATURE:
(D) OTHER Ih-FORMATION: All U's are 2~-F uracil (xi) S~YU~N-~ DESCRIPTION: SEQ ID NO:390:
GGGAGACAAG AAUA~ACGCU CA~NN~NN~N NNNN~NNNNNN NNNNNNNN~N 50 NNNNN~NN NN~NNNNN~ NNNuuC~ACA GGAGGCUCAC AACAGGC 97

Claims (64)

We Claim:

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 a), 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.

5. 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 (2'- NH2) modified ribonucleic acids and 2'-fluoro (2'-F) 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).

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).

10. 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.

15. 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 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.

19. The nucleic acid ligand of Claim 15 wherein said lectin is wheat germ agglutinin.

20. 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 (NH2) 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.

25. 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.

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'-NH2) modified ribonucleic acid and a 2'-fluoro (2'-F) 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.

35. 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).

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.

45. 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'-NH2) modified ribonucleic acid, a 2'-fluoro (2'-F) modified ribonucleic acid, and a 2'-O-Methyl (2'-O-Me) 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 25.

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.

50. 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-selectin as a ligand selected from the group consisting of the sequences set forth in Tables 19, 21 and 25.

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 25.

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 ribonucleic acid.

55. The nucleic acid ligand of Claim 54 wherein said modified ribonucleic acid is selected from the group consisting of 2'-amino (2'-NH2) modified ribonucleic acids, 2'-fluoro (2'-F) modified ribonucleic acids and 2'-O-Methyl (2'-O-Me) modified ribonucleic acids.

56. The nucleic acid ligand of Claim 15 wherein said ligand is a deoxyribonucleic acid.

57. The nucleic acid ligand of Claim 15 wherein said ligand has been further chemically modified at the sugar and/or phosphate and/or base.

58. A multivalent Complex comprising a plurality of ligands of Claim 15.

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 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 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