CA2223003A1 - High-affinity nucleic acid ligands of cytokines - Google Patents

Abstract

Methods for identifying and preparing high-affinity nucleic acid ligands to cytokines and the ligands obtained thereby are disclosed.

Description

W O 96/40717 PCTrUS96/09537 R~G H ~FINITY NUCLEIC ACID LI~.~NDS OF C~rrOKINES

FTF,T.n OF T~ DN~ENTIO N
Described herein are methods for identifying and ~Jie~ g high-affinity nucleic 5 acid ligands to cytokines. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution o!f T ig~n-l~ by Exponential ~nri~hment This invention specifically includes methods for the identification of high affiIuty nucleic acid ligands ofthe following cytokines: IFN-g~mm~ IL-4, IL-l0, TNFa, and RANTES.
Fur~er disclosed are RNA ligands to IFN-p;~mm~ II,-4, IL-l0, and TNFa. Also disclosed are DNA ligands to RANTES. Specific examples are provided of oligonucleotides COIIIH;II;II~ nucleotide derivatives ch~mic~lly modified at the 2'-positions of pyrimi(1inPs The oligonucleotides of the present invention are useful as ph~rm~euticals or diagnostic agents.
l~CKGROUND OF TF~F, TNVF.l~TION
Cytokines are a diverse group of small proteins that mediate cell ~i~n~lin~/comrnunication. They exert their biological functions through specific receptors expressed on the surface of target cells.

2 o Cytokines can be subdivided into several groups, including the immlln~/hematopoietins, hllelre~olls, tumor necrosis factor (TNF)-related molecules, and the chemokines. Re~,~;se"l~liv~ immlmç/hematopoietins include el yll-~ ,poietin (EPO), granulocyte/macrophage colony-stimlll~ting factor (GM-CSF), granulocyte colony-stimlll~tinf~ factor (G-CSF), lellk~mi~ inhibition factor (LIF), oncostatin-M (OSM), 2 5 cilary neuloll-~hic factor (CNTF), growth hor none (GH), prolactin (PRL), interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-l0, and IL-12. Rc~cs~ liv~e interferons (IFN) include IFNa, IFN,B, and IFN-~mm~ R~-iese"lalive TNF family members include TNF ~, i~ reion (IFN) ,B, gp39 (CD40-L), CD27-L, CD30-L, and nerve growth factor (NGF). Represen~li~/e chemokines include platelet factor (PF)4, platelet basic 3 o protein (PBP), groa, MIG, l~NA-78, macrophage infl~mm~tl~ry protein (MIP)l a, MIPl monocyte ~h~mo~ttractant protein (MCP)-l, I-309, HCl4, C l0, Regulated on Activation, Normal T-cell Expressed, and Secreted (RANTES), and IL-8.

CA 02223003 1997-12-0l W O 96/40717 PCT~US96/09537 IFN-gam ma IFN-gamma was first described 30 years ago as an antiviral agent (Wheelock, 1965). Since that time the protein has been shown to be a r~m~rk~hly pleiotropic cytokine which plays important roles in mod~ ting virtually all phases of immllne and 5 infl~mm~tory responses. The cDNAs for murine IFN-gamma (Gray and Goeddel, 1983) and human IFN-gamma (Gray and Goeddel, 1982) have been cloned, sequenced, and char~-~t~ri7~1 IFN-gamma is a member of a family of proteins related by their ability to protect cells from viral infection. This family has been divided into three distinct classes based on 0 a variety of criteria, IFN-alpha (originally known as Type I IFN or Leukocyte IFN), IFN-beta (also originally known as Type I IFN or Fibroblast IFN) and IFN-gamma (ori~in~lly known as Type II IFN or Tmmlme IFN). IFN-gamma is unrelated to the Type I
hll~.r~ s at both the genetic and protein levels (Gray et al., 1982). The human and murine IFN-gamma proteins display a strict species specificity in their ability to bind to 15 and activate human and murine cells. This is due at least in part to their modest homologies at both the cDNA and amino acid levels (60% and 40% respectively).
IFN-gamma is produced by a unique set of stimuli and only by T Iymphocytes and natural killer (NK) cells. The human and murine genes for IFN-gamma are 6 kb in size, and each contain four exons and three introns. These genes have been localized to human 2 o chromosome 12 (12q24.1) and murine chromosome 10. Activation of the hurnan gene leads to the I . ni~ .Lion of a 1.2 kb mRNA that encodes a 166 amino acid polypeptide (Derynck et al., 1982). The human protein contains a 23 residue amino terrnin~l hydrophobic signal sequence which gets proteolytically removed, giving rise to a mature 143 residue positively charged polypeptide with a predicted molecular mass of 17 kDa.
25 Variable post-translational c.~yllla~ic degradation ofthe positively charged carboxy terminl-c (~inrl~rknecht et al., 1984) is responsible for the charge heterogeneity of the fully mature molecule. Proteins with six dirre-~:--l c~l,oxy termini have been detected for both natural and recombinant forms of IFN-g~nnm~ Two polypeptides self-associate to forrn a homodimer with an a~ al~..t molecular mass of 34 kDa (Scahill et al., 1983). The3 0 homodimer is the biologically active form of the protein. Mature human IFN-gamma cc,..L~ins no cysteine residues, thus the homodimer is held together entirely by noncovalent W O 96/40717 PCTrUS96/09537 forces. This qu~t~rn~ry structure of the native protein explains its char~cteri~tic sensitivity to extremes of heat (protein denatured at tempcl~lulcs above 56~), and pH (activity rapidly lost at pH values less than 4.0 and greater than 9.0) (Ml-lkerrin and Wetzel, 1989).
The r~m~rk~ble pleiotropic effects of IFN-gamma are me~ t~d through binding to a single 5 type of IFN-gamma receptor. The structure and function of murine and human IFN-gamnma l~,C~ have been described (Schreiber et al.~ 1992). These receptor proLeins are e ~ ssed on nearly all cells (except erythrocytes), and platelets (Anderson et al., 1982). The receptor binds ligand with high affinity (Kd = 10-9 - 10-l~M) and is essed on most cells at modest levels (200 - 25,000 sites/cell). Upon IFN-gamlma 10 binding to the receptor at the cell surface, the intr~ell~ r dornain of the receptor is phosphorylated at serine and threonine residues (Hershey et a!l.~ 1990).
One of-the major physiologic roles of IFN-gamma is as a regulator of immllne response induction, specifically its ability to regulate ~lession of class I and II major histocomp~tibility (MHC) ~ntigen.e on a variety of immlmologically important cell types 15 (Trinchieri and Perussia, 1985) Functionally, IFN-gamma dependent upregulation of MHC gene e~les~ion is an i.~ oll~ll step in promoting antigen ~rese~ lion during the inductive phase of immune responses and may play a role in ~mtitllnnor activity of IFN-gamma (B~lehm~ier and Schreiber, 1985).
Another major physiologic role for IFN-gamma is its ability to activate human 2 o macrophage ~iy lolo~icity (Schreiber and Celada, 1985). Therefore, IFN-gam~na is the primary cytokine responsible for in~ cing nonspecific cell-me~ te~l me- h~ni.cm~ of host defense toward a variety of intracellular and eYtr~c ~llular ~iles and neoplastic cells (Bancroft et al., 1987). This activation is a result of several distinct functions of IFN-g~mm~ IFN-gamma has been shown to effect the differ,entiation of imm~tllre 2 5 myeloid precursors into mature monocytes (Adams and Hamilton, 1984). IFN-gamma promotes antigen ~l~sellL~lion in macrophages, through the induction of MHC class II
expression as described above, but also by hlcleasing levels of several intracellular enzymes important for antigen processing (Allen and Unanue, 1987). Macrophage cell surface proteins such as ICAM-l are upregulated by IFN-g~m m~, thus enhancing the 3 o functional results of the macrophage-T cell interaction during antigen ~I- ;,ent~Lion W O 96/40717 PCT~US96/09537 (Mantovani and Dejana, 1989). IFN-gamma activates the production of macrophage derived cytocidal compounds such as reactive oxygen- and reactive nitrogen-intt~rmç~ tt?s and tumor necrosis factor-a (TNF-a) (Ding et al., 1988). IFN-gamma also reduces the susceptibility of macrophage populations to microbial infections.(Bancroft et al., 1989).
5 Animal models have been used to study the role of IFN-gamma in the clearance of microbial pathogens. Nelltr~li7ing monoclonal antibodies to IFN-gamma were injected into mice before infecting them with sublethal doses of various microbial pathogens.
These mice lost their ability to resolve the infection initi~ted with Listeria monocytogenes (Bllrhmeier and Schreiber, 1985), Toxoplasma gondii (Su_uki et al., 1988), or Leishmania major (Green et al., 1990).
Besides these nonspecific cell me~ ted cytocidal activities, IFN-gamma also enh~n- çs other macrophage immllne response effector functions. IFN-gamma up-regulates expression of Fc receptors on monocytes/macrophages (FcgRI), thus ~nh~nt~ing the capacity of the macrophage for antibody dependent cell killing (Erbe et al., 15 1990). IFN-gamma also promotes humoral i.n,~;ly through enhancçmel-t of complement activity. It does this in two ways, i) by promoting the synthesis of a variety of complement proteins (ie., C2, C4, and Factor B) by macrophages and fibroblasts, and ii) by regulating the ~ies~ion of complement l~cepto,~ on the mononuclear phagocyte plasma membrane (Strunk et al., 1985).
2 o IFN-gamma also exerts its effects on other cells of the immlme system. It regulates immllnoglobulin isotype switching on B cells (Snapper and Paul, 1987).IFN-gamma plays a positive role in the generation of CD8+ cytolytic T cells (CTLs) (Landolfo et al., 1985) and enh~n~çs NK cell activity. Recently, it has been unequivocally established that CD4+ T cells do not con~LiluLe a homogeneous class of cells. Indeed, a 2 5 paradigm of lymphokine biology and of the function of CD4+ T cells has arisen, the so-called Thl/Th2paradigm (for a review see Paul and Seder, 1994). The THI clones, through their production of IFN-g~mm~ are well suited to induce enh~nred microbicidal and antitumor activity in macrophages as detailed above (enh~n~ed cellular imrnunity), while the Th2 clones make products (IL-4, IL-S, IL-6, IL-10, IL-13) that are well adapted 3 o to act in helping B cells develop into antibody-producing cells (e~h~n~ed hurnoral immunity). The role played by IFN-gam na at this crucial nexus of T cell effector function W O 96/40717 PCTAUS9G/'U53~7 is fi-n(1~ment~1 to the success or failure of the immnn~ response.
IFN-gamma plays a major role in promoting infl~mm~tory responses both directly, and indirectly through its ability to enhance TNF- c~ production. During an infl~mm~tory response, cells leave the circulation and migrate to the point of infection. During this 5 process they must first bind to and then extravasate through vascular endothelium. Both IFN-gamma and TNF-~ can promote the c~icssion of overl~lpping sets of cell adhesion molecules (ICAM-1, E-selectin, and others) that play an important role in this process (Pober ef al., 1986; Thornhill et al., 1991). In fact, ~;x~ e]~ts have shown that these two cytokines exhibit synergistic effects in up-regulating cell adhesion molecules in vivo 1 0 (Munro et al., 1989). One can envision microbial infections iin which the microorganism is already widespread at the time the immlme response develops or in which the response does not quickly rid the host of the infectious agent. This results in continued T cell activation in~ cinp both local infl~mm~tion and tissue damage with Pn~lling loss of normal function. Indeed, when the infectious agent is of little intrinsic pathogenicity, the 15 disease in~ ce-l by the infection may largely reflect the consequences of such a response.
Excessive production of IFN-gamma may play a role in autoi. ~ disorders (forreview see Paul and Seder, 1994 and Stei..ll.~-, 1993). The m~-~h~ni~m for this may involve excessive tissue damage due to aberrant or enh~ncecl ~ ession of class I and class II MHC molecules or the role of an excessive THI cellular response. A role for 2 o IFN-gamma and the tiSsue-~l~m~ging effects of immlm~ responses me~ tecl by T,~,-like cells has been suggested in ~uLoi...-..l...e disorders such as rh~llm~toid arthritis (Fek1m~nn, 1989), juvenile diabetes (Rapoport et al., 1993), my~th~ni~ gravis (Gu et al., 1995), severe infl~mm~tory bowel disease (Kuhn et al., 1993), and multiple sclerosis (Traugott, 1988).

Interleukin-4 (IL-4) is a r~m~rk~bly pleiotropic cytokine first identified in 1982 as a B cell growth factor (BCGF) (Howard et al., 1982). In that same year, IL-4 wasidentified as an IgG1 enhancing factor (Isakson et al., 1982). Because of the effect IL-4 3 o has on B cells, it was first called BCGF-l, later terrned BSF-l (B-cell stimulatory factor-l), and in 1986 it was given the name IL-4. The cDNAs for murine IL-4 (Noma et W O 96/40717 PCT~US~G~'~5;~7 al., 1986; Lee et al., 1986) and human IL-4 (Yokota et al., 1986) have been cloned, sequenced, and chara~L~ d.
IL-4 can be regarded as the ~lol~Ly~lic member of a family of immune recognition-inrlllced lymI-h~ kinPs de~ign~ted the "IL-4 family" (for a review see Paul, 1991). This family consists of IL-4, IL-5, IL-3, and granulocyte-macrophage colony-stim~ tin~ factor (GM-CSF). The p~ .Lies shared by these p-~teills leads to this grouping and include, i) the linkage of the genes for the members of the family (van Leeuwen et al., 1989), ii) the action of each member of the family as a hematopoietic growth factor in addition to any effects it rnay exert on lymphoid cells, iii) the receptors 1 0 for these proteins are all memhers of the hematopoietin family of lece~lol~ (Bazan, 1990a), and iv) coexpression of these factors by a subpopulation of cloned CD4+ T cells (the so-called TH2 cells) (Mosmann et al., 1989) and by mast cells (Plaut et al., 1989).
The r~m~rk~hle pleiotropic effects of IL-4 are me~ tP~l t_rough binding to cell surface receptors (IL-4R). The murine IL-4R (Mosely et al., 1989; Harada et al., 1990), and the human IL-4R (Idzerda et al., 1990; Galizzi et al., 1990) have been cloned, sequenced, and characterized. IL-4R are present on a variety of hematopoietic (Park et al., 1987) and nonhem~topoietic cells (Lowenthal et al., 1988). On both human and murine resting T and B cells, IL-4R are present in low numbers (<400) and are regulated by cytokines and other factors. The receptor binds IL-4 with high affinity (Kd = 10-'~ M).
o Now that most of the receptors for imml-noregulatory and hematopoietic cytokines have been cloned, it is ~ nl that the majority of these l~ec~Lol~ fall into a large family. This hematopoietic cytokine receptor ~u~,r~l-ily includes receptors for IL-4, IL-2 ( b and g chains), IL-7, IL-9, and IL-13 which modulate the lymphoid system; and receptors for cl~L~Ilo~oietin, granulocyte-colony stim~ ting factor (G-CSF), GM-CSF, IL-3, and IL-5 2 5 which modulate the hemopoietic system. The superfamily also includes receptors for factors believed to normally function outside the immlm~ and hematopoietic systems, including receptors for growth hormone (GH), prolactin, leukemia inhibitory factor (LIF), IL-6, IL-11, and ciliary neurotrophic factor (CNF) (for a review see Kishimoto et al., 1994).
3 o A general first step in the sign~ling processes of immune and hematopoietic cytokines may be ligand-in~luced di~l" .i~Lion of receptor components whose cytoplasmic W 0 96/40717 PCT/U'r~'~9537 regions interact to initiate a dow~ ea-~l ci~n~lin~ c~cc~ Tlhe IL-4 receptor has a long putative intr~qce~ r domain (553 amino acids in mouse, 569 in human) with no known con~n~ sequences for kinase activity or for nucleotide-binding regions. The biochemical nature of signals in~ cetl by the binding of IL-4 to its lec~l(,r have not been 5 elucidated. It does appear that the cytosolic domain of the receptor is essential for its .ci~n~lin~ function (Mosely et al., 1989). Ligand in(1l1red ~limeri7~tion of the IL-4 receptor appears to be a critical first step in IL-4 mediated signal tr~n~ tion.
One of the major physiologic roles of IL-4 is as a B lymphocyte activation and di~~ ,liation factor (Rabin et al., 1985; Oliver et al., 1985). The protein was first 0 isolated based on this activity. In this regard, IL-4 activates production of IgGl (Vitetta et al., 1985), but is also responsible for isotype ~wil;hh~g in B cells from production of IgG
to IgE immllnl~globulins (Coffman et al., 1986; Lebman and ~Coffman, 1988, Del Prete et al., 1988). The effect of IL-4 on the in vivo regulation of IgE has been clearlydemonstrated. Neutralization of IL-4 by tre~tm~nt with a monoclonal anti-IL-4 antibody 15 (Finkelm~n et al., 1986) or a monoclonal antibody to the IL-4 receptor (Finkelm~n et al., 1990) will block the IgE response. A recombinant soluble IL-4 receptor has been shown to inhibit IgE production by up to 85% in vivo (Sato et al., 1993). IL-4 deficient mice produced by gene-l~tillg in murine embryonic stem cells have normal B and T celldevelopment, but serum levels of IgGl and IgE are strongly reduced (Kuhn et al., 1991).
2 o IL-4 ~llgment~(l IgE production leads to an atopic state (allergy/asthma) (Finkelman et al., 1989; Katona et al., 1991).
The IL-4 mediated up-regulation of IgGl may play a role in the infl~mm~tion c~c~-le IgGl has recently been shown to form ;~ e complexes which bind to the cellular leceptc ls for t]he Fc domain of immlln~globulins (FcRs) leading to an 25 infl~mm~tr~ry response (Sylvestre and Ravetch, 1994; Ravetc]h, 1994). IL-4 transgenic mice have been produced that hyperexpress IL-4 (Tepper et al., 1990). These mice have elevated levels of serum IgGl and IgE and they develop allergic infl~mm~tory disease.
These findings demonstrate the critical role IL-4 plays in the humoral imml-n~ response.
Another major physiologic role for IL~ is as a T Iymphocyte growtlh factor (Hu-Li 3 0 et al., 1987; S pits et al., 1987). IL-4 enhances the proliferation of precursors of cytotoxic T cells (CTLs) and their differentiation into active CD8~ CTLs (Widmer and Grabstein, 1987; Trenn, 1988). IL-4 appears to ~llgment the IL-2 driven induction of lymphokine-activated killer (LAK) cells (Higuchi et al., 1989), which have been shown to lyse a variety of tumor cell targets without major histoco~ aLibility complex (MHC) restriction. The role played by IL-4 at this crucial nexus of T cell effector function is filn~ m~nt~1 to the success or failure of the immlln~ onse.
IL-4 has been shown to affect nonlymphoid hematopoietic cells in a variety of ways. IL-4 has been shown to modulate monocyte/macrophage growth (McInnes and P~nnick, 1988; Jansen et al., 1989) while enh~n~ing their dirr~ iation and cytotoxic activity for certain tumor cells (Crdwrol-l,et al., 1987; Te Velde et al., 1988). IL-4 also 0 has activity as a stim~ nt of mast cell growth (Mosmann et al., 1986; Brown et al., 1987), and increases production and re~cruitment of eosinophils (Tepper et al., 1989).
IL-4 alone or in conju~ lion with other cytokines can promote the expression of variety of cell-surface molecules on various cell types with diverse implications for e~e Specifically, IL-4 can interact with tumor necrosis factor (TNF) to selectively enhance vascular cell adhesion molecule-l (VCAM-l) ~r,~ ion contributing to T cell extravasation at sites of infl~mm~ti~n (Briscoe et al., 1992). IL-4 alone or in combination with TNF or IFN-gamma has been shown to increase both MHC antigen and tumor-associated antigen t;~,es~ion on a variety of neoplastic cells (Hoon et al., 1991).
As ~iet~ile-l above, IgGl immune complexes bind to the cellular receptors for the 2 o Fc domain of immlln~globulins (FcRs) leading to an infl~mm~tory response. Inhibition of IL-4 and the subsequent reduction in IL-4 meAi~te~ IgGl ex~ ion may prove efficacious against immlm~ complex infl~mm~tory disease states. Indeed, inhibitory ligands to IL-4 might also prevent the IL-4 m~ tPd o~ ;x~.e3sion of VCAM-l, thuss~tt~nn~tin~ the ability of T cells to extravasate at sites of infl~mm~tion.
2 5 Inhibition of IL 4 activity with a monoclonal antibody, a recombinant soluble IL~
~celltor, or gene knock-out, results in a reduction of serum IgE levels. An inhibitory oligonucleotide ligand to IL 4 could be clinically effective against allergy and allergic s~thm~
A recent report has described a disorder in bone homPost~ci~ in transgenic mice 3 o that inal.l.,ul,.;ately express IL~ under the direction of the Iymphocyte-specific proximal promoter for the Ick gene (Lewis e? al., 1993). Bone disease in these mice resulted from CA 02223003 l997-l2-Ol W O 96/40717 PCTrUS~G~'~5~7 markedly decreased bone formation by osteoblasts, ft;~Lu~es identical to those found in human osteoporosis. Inhibiting this IL-4 mediated reduction in osteoblast activity may ~ prove beneficial against osteoporosis.
Graft-versus-host disease (GVHD) is a major complication of human tissue tr~n~pl~nt~fion. GVHD does not exist as a single clinical manire~lalion but can involve immlmc-logic abnormalities ranging from immlmodeficiency to systemic autoi""~ es(Ferrara et al., 1991). These systemic auto;...,..~ ;es include clinical and serological manifest~tion~ of human systemic lupus erythl?m~tosus (SLE). Several murine models of SLE have been developed (Gleichm~nn et al., 1982; van Rappard-van Der Veen et al., 1982), and the induction of systemic GVHD in mice has been described (Via et al., 1988).
Two recent studies have shown in vivo efficacy of a mouse monoclonal antibody to IL-4 in preventing GV- HD and SLE in these murine model systems (IJmland et al., 1992, Ushiyama et al., 1995). These obs~l~/alions suggest that an inhibitor of human IL-4 may be effective in tre~tment of chronic systemic auloi..----l-.-iLies such as SLE and GVHD.
A variety of microbicidal infections are rh~r~ct~rized Iby depressed cellular but enhanced humoral immlm~ responses, which suggests a TH2 type of response to infecti~n This TH2phenotype is ch~r~cteri7Pd by T cell secretion of IL~, as detailed earlier. IL-4 blocks the microbicidal activity of IFN-gamma activated macrophages in fiphting Leishmania major infection (Liew et al., 1989, Leal et al., 1993). Inhibition of IL-4 2 o would ~nh~n-~e the THI effector arm of the immnn~ response enhancing cellular immtlnity and leading to the resolution of infection. Neutralization of IL-4 in vivo allows mice otherwise susceptible to Leishmania major infection to fight offthe parasite and clear the infection (EIeinzel et al., 1989). Several inform~tive studies have looked at the THl/TH2 phenotypic ~ tint~tion in infected mice, and suggest a THI ~lomin~t~rl re~ollse being most 2 5 effective in fightin~ microbial infection (for a review, see Sher and Coffman, 1992).

W O 96/40717 PCT~US96/09537 IL-10 is a cytokine produced by the Th2 cells, but not Thl cells, and inhibits synthesis of most of all cytokines produced by Thl cells but not Th2 cells (Mosmann et al., 1991). In addition to the effect on CD4+ cells with Thl phenotype, IL-10 also inhibits 5 CD8+ T cells with "Thl-like" phenotype. IL-10 is a potent suppressor of macrophage activation. It can ~,u~less the production of proinfl~mm~tory cy+~okines, including TNFa, IL-1, IL-6, IL-8 and IFN-g~mm~ Overall, these results suggest that IL-10 is a potent macrophage deactivator and an effective anti-infl~mm~tory reagent. In addition, IL-10 plcvl;llL~ the IFN- g -inclllce~l ~yllllle.,is of nitric oxide, resl-ltin~ in decreased resict~nce to 10 intracellular ~ ,iLes (G~77in~11i et al., 1992).
Both human and mouse (hIL-10 and mIL-10, respectively) have been cloned and expressed (Moore et al., 1990; Vieira et al., 1991). The two cDNAs exhibit high degree of nucleotide sequence homology (>80%) throughout and encode very similar open reading frames (73% amino acid homology). Both plvteills are expressed as noncovalent 15 homodimers that are acid labile (Moore et al., 1993). Whether monomers are equally bioactive is not clear yet. Based on the ~l;lll~/ structure IL-10 has been categorized into the four a-helix bundle family of cytokines (Shanafelt et al.,l 991). Possibly due to high degree of sequence homology and similar structure hIL-10 has been shown to be active on mouse cells (Moore et al., 1993) but not vice versa. hIL-10 is an 18 kDa polypeptide with 2 o no ~letect~hle carbohydrate, however, in mIL-10 there is one N-linked glycosylation. The recombinant hIL-10 has been ex~lessed in CHO cells, COS7 cells, mouse myeloma cells, the baculovirus t;x~res~ion system and E. coli. The rIL-10 ~x~lc;i,sed in these systems have inclietinpuishable biological behavior (Moore et al.,l993).
Parasitic infection often leads to polarized immllne response of either Thl or Th2 2 5 type which can mediate protection or susceptibility. The outcome of a parasitic infection depends on the nature of the parasite and the host. The best understood example is Leishmania major infection in mice. L. major is a protozoan parasite that establishes an intracellular infection in macrophages, where it is mainly localized in phagolysosomes.
Activated macrophages can efficiently destroy the intracellular parasite and thus parasitic 3 o protection is achieved by macrophage activation. Nonactivated macrophages do not kill these org~nicmC As expected. activation of macrophages upon IFN-gamma tre7~tm~nt W O 96/40717 PCT~US96/09537 ~onhzmf e~l the protection, wherea,s IL-4 a~nd IL-10 blocked the increased microbicidal activity in~ ce~ by IFN-gamma (Liew et al., 1989). In most inbred strains (example, C57/BL6) c~-t~n~ous infection of L. major often leads to localized infection with spontaneous healing a~nd confers rÇ~i~t~nre to rehllè~;lion. HoweYer, in BALB/c mice, L.
major infection in~ es nonprotective immtm~ response by producing IL-4. The antibody response m~ tecl by IL-4 is ine~e-;live and leads to death (Howard et al., 1980). In healing strains a strong Thl l~syollse has been noticed with high level of IFN- g, whereas in susceptible BALB/c mice a nonproductive Th2 response with siEnifi~nt levels of IL-4 was found (Heinzel et al., 1991). Further it was shown that a~ single injection of monoclonal anti-IFN-gamma antibody can convert a re~i~t~nçe into a susceptible mouse (Belosevic et al., 1989). As expected, the tre~tm~nt of BALE~/c mice with anti-IL-4 a~ntibody led to the development of Thl response alnd healing (Sher & Cof~llan, 1992).
Thus, depending on the nature of the pathogen, ~h~nging the immlm~ response to a T cell subset with a ~lole-;live phenotype can lead to th~,.d~ ~lic intervention of the disease state.
Underst~n-lin~ the regulation between the Thl and Th2 phenotype m~ tçcl by cytokines will help in clç~i ning cytokine-antagonist in thcldyculics. T]he production of IL-10 is strongly increased in mice infected with various pathogens such as Leishmania major, Schistosoma mansoni, Trypanosoma cruzi and Mycobacterium Leprae (Sher et al, 1992;
~lp;,~me et al., 1991, Heinzel et al., 1991).
2 o When ~lçci ning immlme therapy to facilitate mounting the right arrn of defense me~h~ni~m toward pathogens, it is hnyull~ll to ~ iu a balance between the two arrns also. Th2-type responses may be hlly~ ll in controlling the tissue damage m~ tç~ by Thl cells during the response to an intracellular infectious agent. Keeping some Thl cells functioning in a predo---il~--lly Th2 c~ ol~nent can help abrogate (l~m~ging effects of Thl by secreting IL-10 and IL4. One extreme ofthe spectrum of Thl/Th2 is reflected in transgenic mice lacking the IL-10 gene (Kuhn et al., 1993). The IL-10 deficient mouse is normal with respect to its development of T and B cell subsets. However these mice develop chronic enterocolitis (or infl~mm~tory bowel disease) due to chronic infl~mm~tion via continuous overyroduction of cytokines such as TNFa and 3 o IFN-g~mm~(Thl response).

IL-12 can also induce the development of the Thl subset. By using Lysteria monocytogen, an intracellular grarn-positive bacteriurn, infection in antibody T cell receptor transgenic mice as a model it has been shown that IL-10 can block the production of IL-12 from macrophages (Hsieh et al.,1993) . Thus an IL-10-antagonist will tip the 5 Thl/Th2 population predo~ ly to Th2 type environrnent by 1, ~l~venlillg the inhibition of the production of Thl cytokines 2 by allowing the production of a cytokine that intlllc~s the development of Thl subset.
With exp~riment~l evidence in hand it has been proposed that the rçeiet~nre and/or progression to AIDS is dependent on a Thl/Th2 stage of an individual (Clerici & Shearer, 10 1993). This hypothesis is based on the fintlinge that progression to AIDS is characterized by loss of IL-2 and IFN-gamrna production (loss of Thl response) with increase in IL-4 and IL-10 (acquired Th2 response). Many seronegatives (HIV-exposed individuals) generate a strong Thl-type response. It is important to note that after seroconversion both IL-4 and IL-10 levels go up at the ~x~ense of IL-4 and IFN-gslrnm~ However, in l 5 full-blown AIDS p~ti-qntc, Th2 response seems to be m~ t~cl by high levels of IL-10 but not with IL-4, the level of which goes down to norrnal in these individuals. An anti-IL-10 reagent may serve as a potential th~.d~;uLic in shifting the Th2 response to Thl in AIDS
patients to offer protection.

TNFa TNFa is an extracellular cytokine and a central mediator ofthe immnn~ and infl~mm~tory response (Beutler et al., 1989; Vassalli, 1992). It is a homo-trimer (Smith et al., 1987, Eck et al., 1988), and has a subunit size of 17 kD. It circulates at concentrations of less than 5 pg/ml in healthy individuals (Dinarello et al., 1993) and it can go as high as 25 1000 pg/ml in patients with sepsis syndrome (Casey et al., 1993). The human TNFa is nonglycosylated, whereas in some other species (notably the mouse) glycosylation occurs on a single N-linked site in the mature protein, but the sugar moiety is not ecsenti~l for biological activity (Beutler et al., 1989). The human TNFa is acidic with a pH of 5.3 (Aggarwal et al., 1985). Each TNF~ subunit concictC of an anti parallel l~-sandwich and it 3 o participates in a trimer formation by an edge-to-face p~CIcing of ~sheets. The structure of the TNFa trimer resembles the "jelly-roll" structural motif characteristic of viral coat W O 96/40717 PCT~US96/09537 plol~ills (Jones et al., 1989). TNFa is a relatively stable molecule and may be exposed to chaotropic agents such as urea, SDS, or gll~niclinium hydrochloride, and renatured with recovery of as much as 50% of the initial biological activity. The TNF~ renaturability may reflect the limited number of int~rn~ fi~e bonds (one per monomer) required for 5 ".~;..tf ~ ce of structure (Beutler et al., 1989).
Another related molecule, TNF~3, has the same bioactivity as TNFa . The "lkL~e~;ies sequence identity within the TNFa and TNF~ families is 71% and 61%, respectively (Beutler et al., 1989). The sequence identity betwee,. hTNF-~ and hTNF-~3 is only 29% (Beutler ef al., 1989). Despite their low ~imil~rity, both hTNFa and hTNF~3 0 bind to the same ,~ c~Lv,~ with eo, . ~ ble ~ffinities TNFa m~ tPc its bioactivity through binding to cell surface receptors. The TNFcc receptors are found on the surface of virtually all somaLtic cells tested (Vassalli, 1992). Two distinct TNF~ lect;~to,s have been char~cteri7~d of ~lJ~e.ll molecular weights 55kD (p55 TNFa-Rl) and 75kD (p75 TNFa-R2) (Hohmann etal., 1989;
5 Brockhaus et al., 1990; Loetscher et al., 1991). Both recepto;rs bind TNFa and TNF~3 with high ~ffini~i~s (Kd=0.3-0.6 nM) (Loetscher et al., 1990; Schall et al., 1990; Per~nica et al., 1992).
TNF~ has diverse activities, and thus is implicated in several tli~e~es as follows:
Septic shoc~ Sepsis incidents have been inc"~asil,g for thLe last 60 years and is the 2 0 most cornmon cause of death in intensive care units in the United States (Parrillo, 1991).
The mortality of septic shock remains at a~ploxhl-ately 50% despite the standard use of ag~ s~i~e antibiotics and cardiovascular support for the past 10 years (Parrillo, 1991).
The evidence implicating TNF~ in sepsis is as follows. Pl~;L,e~l~..ent of mice or baboons with monoclonal antibodies to TNFa protects them from lethal doses of E. coli LPS
2 5 (Beutler et al., 1985). Anti-TNFa antibodies protect primates against lethal endotoxin sepsis and against lethal S. aureus-int~ e~l shock (Fiedler et al., 1992; Hinshaw et al., 1992). Soluble-TNFa-receptor (pS5)-IgG-Fc fusions (INFa receptor immuno~-~h~sin)- were found to protect mice from endotoxic shock, even when ~r1mini~tered lhr after endotoxin infusion. The same immlmo~-lh~cin was also effective against listeriosis in 3 0 mice (Haak-Frendscho et al., 1994). Another immlmoadhesin based on the p75 receptor W O 96/40717 PCTrUS96/09537 was also shown to be effective in lethal endotoxemia and it was functioning eimlllt~neously as both TNF~ carrier and TNFa antagonist (Mohler et al., 1993).
Cachexia In vivo ~lminietration of TNFa causes c~chP~i~ in mice (Oliff et al., 1987). Therefore, TNFa antagonists may protect cancer or AIDS infected patients from 5 c~ch~
Cerebral malaria High levels of TNFa are associated with poor prognosis in children with cerebral m~l~ri~, and antibodies to TNF a protect mice from cerebral complications of Plasmodium berghei infection (Grau et al., 1987).
Arthritis. Antibodies to TNFa reduce the production of the infl~mm~tory 10 cytokine, IL-l in synovial cells (Brerman et al., 1989). TNF a is an inducer of collagenase, the major destructive protease in rh~llm~toid arthritis (Brennan et al., 1989).
Anti-TNF a antibodies were found to ameliorate joint disease in murine collagen-in~lnre-l arthritis (Williams et al., 1992). Transgenic mice carrying the hTNFa gene develop arthritis which can be prevented by in vivo ~1minietration of a monoclonal antibody 5 against hTNFa (Keffer et al., 1991).
Graft Rejection and Graft versus Host Reaction (GVHR). TNFa has been implicated in the acute phase of graft-versus-host disease and in renal allograft rejection.
Antagonists of TNFa may then be able to prevent these life-tllle ~ ~ e~ . i, .g conditions.
Anti-TNFa antibodies have been found to delay graft rejection in t~ llental ~nim~l~
2 o (Piguet, 1992). Also, injection of anti-TNFa antibodies during the acute phase of GVHR
reduces mortality, and the severity of intestin~l, epid~rm~l, and alveolar lesions (Piguet, 1992). Clinical trials of the efficacy of anti-TNFa antibody in human bone marrow transplantation are underway.
AIDS. Studies of intracellular signal tr~n~ lrtion ~lllw~y~ revealed that TNFa 2 5 induces proteins that bind to kB-like enhancer elements and thus takes part in the control of NF-kB-inducible genes (Lenardo et al., 1989; Lowenthal et al., 1989; Osborn et al., 1989). The antiviral activity of TNFa at least in part is m.odi~tt-d by the interaction of NF-kB with a virus-inducible element in the ~-hll~. r~.Ol. gene (Goldfeld et al., 1989;
VisvA~ h~ et al., 1989). By an analogous ...P~h~ m, TNFa appears to activate human 3 o immlm~deficiency virus type I (Duh et al., 1989; Folks et al., 1989). Therefore TNFa CA 02223003 l997-l2-Ol W O 96/40717 PCTrUS96/09537 antagonists may prove useful in delaying the activation of the AIDS virus and may work in conjunction with other tre~tment~ in the cure of AIDS.
Parkinson's disease. Recently, elevated TN~a levels have been found in the brainand the cerebrospinal fluid of Parkinsonian patients (Mogi et al., 1994). This report speculates that elevated TNFa levels may be related to neuronal dege~ dLion associated with the tli~e~e 1~ ~NT~.S
RANTES is a small (MW 8-kD) highly basic (pI~9.5) chemokine that belongs to the CC group (Schall, 1991; Baggiolini et al., 1994). It does not appear to be glycosylated (Schall, 1991) and is a chemo~ttractant for monocytes (Schall et al., 1990; Wang et al., 1993; Wied~rrn~nn etal., 1993), basophils (Bischoffetal., 1993; Kunaetal., 1993), eosinophils (Rot et al., 1992), and CD4+/UCHLl+T lymphocytes which are thought to be i.n~ tecl or primed helper T cells involved in memory T cell function (Schall et al., 1990). RANTES is not orlly a chemoattractant but it also stim~ tes cells to release their effectors leading to tissue damage. For example, RANTES c~wses hi~t~rnintq release from basophils (Kuna et al., 199~; Kuna et al., 1993; Alam et al., 1993). It also causes the secretion of eosinophil basic peptide (Alam et al., 1993) and the production of oxygen free radicals (Rot et al., 1992) by eosinophils.
2 0 Initially, it was thought that RANTES was synth~ ocl by activated T cells but recently other cells were found to synthlo~i7~ it very fast upon stim~ tion. RANTES
rnRNA is expressed late (3 to 5 days) after activation of resting T cells, whereas in fibroblasts, renal epithelial and m.?~n~i~l cells, RANTES mRNA is quickly up-regulated by lNFa stim~ tion (Nelson et al., 1993).
2 5 Receptors for RANTES have been identified. There is a promiscuous receptor on the surface of erythrocytes that binds all chemokines with a Kd=5nM (Horuk et al., 1993;
Neote et al., 1993). This receptor is thought to be a sink for chemokines to help in the establi~hment of chemotactic gradients. Signal transducing receptors have also been identified and cloned (Gao et al., 1993; Neote et al., 1993; Van-Riper et al., 1993; Wang 3 o et al., 1993). Mono~ytes carry a G-protein coupled receptor that binds RANTES with estim~te~l Kd of 400 pM, but also MCAF and MIP-la with lower affinities (estim~t~l Kd CA 02223003 l997-l2-Ol W O 96/40717 PCTAUS96/09~37 of 6 and 1.6 nM respectively) (Wang et al., l 993). A receptor molecule has been cloned from neutrophils that can bind RANTES with a lower affinity of about 50 nM (Gao et al., l 993). A
Disease State. RANTES antagonists may have thc.d~uLic application in 5 infl~mm~ti~n. Blockage of the çh~mo~qttractant and ~e~;~ol cell activation prop~ llies of RANTES would block local infl~mm~tion and tissue damage. The mech~ni~m of actionof the RANTES antagonist will be the inhibition of RANTES binding to cell surface ec~ol~.
RANTES is f hemoiqttractant for monocytes, basophils, eosinophils and memory 10 lymphocytes. Basophils are the major source of mediators such as hict~mine and peptido-leukotrienes, and are an eSsenti~l element of the late-phase responses to allergens in lly~ ensitivity ~ e~ces These cells are also involved in other infl~mm~tory pathologies, including certain auto;.,.."l...e reactions, parasitic infections and infl~mm~tory bowel ~ e~ees. In these conditions, basophil le~ "ent and activation is independent 15 of IgE. Numerous reports have ~- c~lmnl~te~l over the years that describe the effects of a group of elusive stimuli operationally called "hi~t~mine releasing factors." A large number of these elusive stimuli may well be contributed by RANTES.
Eosinophiles also are important in allergic infl~m~tion, and together with lymphocytes, form ~lolllillellt infil~tes in the bronchial mucosa of patients with ~thm~
2 o They are believed to be the cause of epithelial damage and the char~ctPri~tic airway hyper-reactivity. The recrllitement of lymphocytes of the Th2 type, which comigrate with eosinophiles into sites of late-phase reactions, is an important source of otherchemo~ttr~-~t~nt cytokines and growth factors that prime eosinophils.
RANTES, with its effects on monocytes, basophils, eosinophils and lymphocytes 2 5 appears to be a potent stimulator of effector-cell ~cc~lmul~tion and activation in chronic infl~mm~tory ~ e~ec and in particular, allergic infl~mm~tion.
The recrllitem~?nt system of infl~mm~tory cells has some recl-m~ncy built into it.
However, RANTES has some unique ~lope-Lies. It is a more potent chemoattractant than MCP-l and MIP-l a, while MCP-l is more potent stimulator of hi~t~minP release from 3 o basophils (Baggiolini et al., 1994). RANTE,S causes the production of oxygen radicals by eosinophiles while MIP-l a cannot (Rot et al., 1992). RANTES is as potent as C5a in the CA 02223003 l997-l2-Ol W O 96/40717 PCTrUS96/09537 . 17 ent of eo~inrhiles, but not as potent a trigger of the eosinophil oxydation burst (Rot et al., 1992). C5a is a very potent chemo~ttractant: however, it lacks the specificity of RANTES. It attracts not only basophils and eosinophils but also n~;uLl~hils. Since the eosinophils, but not the nc;ul~ ils, are hn~ ~l in the pathophysiology of some 5 infl~mm~tory conditions, such as the allergen-in~ ce~l late-phase reaction and asthma, specific çhemo~ ;l~L~ such as RANTES are expected to be involved.
Using in situ hybridization, RANTES e~ ,ssion has been found in hll~ lilial mononuclear cells and proximal tubular epithelial cells in human kidney transplants undergoing rejection. Antibody staining revealed the presence of RANTES not only10 wit_in the i.~ l infiltr~te and renal tubular epithelial cel]s but also in high abundance in inflamed endothelillm (Wie~ et al., 1993). Based on these results a haptotactic mechar~ism was post~ te~l Haptotaxis is defined as cell migration int1uced by surface-bound r~tlientc The haptotactic m~-~h~ni~m was ~u~ ed by in vitro experiments and anti-RANTES antibodies have been found to prevent ~at in vitro 5 haptotaxis.
Human rh~llnn~toid synovial fibroblasts express mRNA for RANTES and IL-8 after stim~ tion with TNFa and IL-113 (~th~n~wami et al., 1993). There is a di~,e~lLial regulation of ~ s~ion of IL-8 and RANTES mRNA. Cycloheximide enh~n~ecl the mRNA levels for IL-8 and RANTES after stim~ ti~n with IL-113 but 2 o reduced the levels of RANTES mRNA after stimnl~tion with TNF~. Also, IL-4 down-regulates and IFN-gamma çnh~n~es the TNFa and IL-113 inrl~lce~l increase inRANTES mRNA, whereas the induction of IL-8 mRNA by TNFa or IL-lJ3 was inhibited by IFN-gamma and ~ngmçnted by IL~. Moreover, the combïnation of TNFa and IL-113 synergistically increased the level of IL-8 mRNA, whereas under the same conditions, the 2 5 levels of RANTES mRNA were less than those intlnced with TNFa alone. These studies suggest that the synovial fibroblasts may participate in the ongoing infl~ nm~tc)ly process in rh.onm~tQid arthritis, and RANTES might be one of the parl:icipating effectors. The observed differential regulation of IL-8 and RANTES indicates that the type of cellular - infiltrate and the progress of the infl~mm~tory disease is likely to depend on the relative 3 0 levels of stiml-l~toly and inhibitory cytokines.

W O 96/40717 PCT~US96/09537 RANTES has also been implicated in atherosclerosis and possibly in postangioplasty restenosis (Schall, 1991). The participation of MCP-l in atherosclerosis has been studied to a greater extent. Recently mRNAs for RANTES, MIP-l a and MIP-l~ have been detected in i~i~ in normal carotid plaque and heart transplant 5 atherosclerosis. RANTES mRNA is not detected in the same cells expressing MIP-la and MIP-113, but it is t;x~ ,ss~d in lymphocytes and macrophages typically more ~ xil"al to the lumen. The data argue for positive feed-back mel~h~nicm~ for the CC chemokines and possible dirrel~,.llial ~le~sion of these chemokines at various stages in the progression of arterial ~lice~ce Finally, elevated RANTES levels have been correlated with endometriosis (Khorram et al., 1993). RANTES levels were elevated in pelvic fluids from women with endometriosis, and these levels correlate with the severity of the ~lice~ce Protein Homology between Human and Animal. The murine RANTES has been cloned (Schall et al., 1992). Sequence analysis revealed 85% amino acid identity between 15 the human and mouse proteins. The human and murine RANTES exhibit imml-n~
crossreactivity. Boyden chamber chemotaxis ~ ;...entc reveal some lack of species specificity in monocyte chemo~LLla~;L~ll potential, as recombinant muRANTES attracts human monocytes in a dose-dependent fashion in vitro. Also, hRANTES transfection into mouse tumor cell lines produce tumors in which the secretion of hRANTES by those2 o tumors correlates with increased murine monocyte infiltration in vivo (Schall et al., 1992).

SELEX
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of 25 T,i~nrlc by EXponential enrichment, termed SELEX, is described in United States Patent Application Serial No. 07/536,428, entitled "Syst~Pm~tic Evolution of T.ig~n~ls by ExponPnti~l EnrichmPnt," now abandoned, United States Patent Application Serial No.
07/714,131, filed June 10, 1991, entitled "Nucleic Acid ~.ip,~ntlc," United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled "Nucleic Acid3 o ~ .ig~nrlc," now United States Patent No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically inco.~,olated by reference. Each of these applications, W O 96/40717 PCT~US96/09537 collectively referred to herein as the SELEX Patent Applications, describes a filnri~m~ t~lly novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a lllixLule of.candidate 5 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 ll~i~lu~'~ of nucleic acids, preferably comprising a se~Tnent of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound 1 0 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-çnri~h~ll mixture of nucleic acids, then 1~ the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the 15 target molecule.
T~e basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Applir~ti~ n Serial No. 07/960,093, filed October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure,"
describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid 2 o molecules with specific structural charact~n~tirc, such as benl~ DNA. United States Patent Application Serial No. 08/123,935, filed September 17, 1993, entitled "Photoselection of Nucleic Acid T ig~ntl~" describes a SELEX based method for selecting nucleic acid ligands cont~ining photoreactive groups capable of binding and/or ph~tocro~linking to and/or photoinactivating a target molecule. United States Patent Application Serial No.25 08/134,028, filed October 7, 1993, entitled "High-AffinityNucleic Acid Ligands That Disclh~ le Between Theophylline and Caffeine," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25, 1993, entitled "System~tic Evolution of T.ig~nr1c by EXponential Enrichment:
3 0 Solution SELEX," describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low af~mity for a target molecule.

United States Patent Application Serial No. 07/964,624, filed October 21, 1992, entitled "Methods of Producing Nucleic Acid T.ig~ntl~" describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. United States Patent Application Serial No. 08/400,440, filed March 8, 1995, entitled "Systematic Evolution of T ig~n~l~ by 5 EXponential Enriçhment- Chemi-SELEX," describes methods for covalently linking a ligand to its target.
The SELEX method enco. ~ es the ic1t?ntific~tinn of highaffinity nucleic acid ligands co~ modified nucleotides conferrin~ improved characteristics on the ligand, such as illl~lvved in vivo stability or improved delivery char~ctt?ri~tics. Examples of such o modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands CO..~ g modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8, 1993, entitled "High Affinity Nucleic Acid T i~nfl~ Co..~ il-g Modified Nucleotides,"
that describes oligonucleotides co..l;1;..;..~ nucleotide derivatives chemically modified at 5 the 5- and 2'-positions of pyrimi(1in~s. United States Patent Application Serial No.
08/134,028, supra, describes highly specific nucleic acid ligands co--l~i--illg 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 Pl~p~dlion of 2' Modified Pyrimi~linP Intramolecular 2 0 Nucleophilic Disp~ ment~ describes oligonucleotides col.l Siil-i .~g various 2'-modified .
pyrlmldmes.
The SELEX method encomp~e~çs combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide fimctional units as described in United States Patent Application Serial No. 08/284,063, filed August 2, 1994, entitled 2 5 "System~tic Evolution of T .ig~n-lc by Exporl~nti~l Fnrichment- Chimeric SELEX" and United States Patent Application Serial No. 08/234,997, filed April 28, l 994, entitled "Sy~l~ ..nl;c Evolution of T.ig~n-lc by Exponential Enri~h...- - l Blended SELEX,"
e~ ely. These applications allow the combination of the broad array of shapes and other plopel Lies, and the efficient amplification and replication ~.o~ c. Lies, of 3 0 oligonucleotides with the desirable propcl Lies of other molecules. Each of the above CA 02223003 l997-l2-Ol W O 96/40717 PCTrUS96/09537 described patent applications which describe modifications of the basic SELEX procedure are specific~lly incorporated by reference herein in their entirety.

RI~Tl;'~ SUMMA~ OF T~TF ~ TION
The present invention includes methods of identifying and producing nucleic acidligands to cytokines and the nucleic acid ligands so identified and produced. In particular, RNA sequences are provided that are capable of binding specifically to IFN-g~mm~ IL-4, IL-10, and TNFa. In addition, DNA sequences are provided that are capable of binding specifically to RANTES. Specifically included in the invenl:ion are the RNA ligand sequences shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73; 79-185; 189-205;
209-255).
Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to a cytokine comrri~ing 1he steps of (a) ~l~,~h~g a candidate ~ e of nucleic acids, (b) col t~eting the c~n~ te nlixlulc; of nucleic acids with a cytokine, (c) partitioning between members of said c~ln-lirl~t~ on the basis of affinity to the cytokine, and (d) amplifying the selected molecules to yield a lnixlule of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to the cytokine.
Further included in this invention is a method of identifying nucleic acid ligands 2 o and nucleic acid ligand sequences to a cytokine selected from the group con~i~ting of IFN-p;,.mm~, IL-4, IL-10, TNFoc, and RANTES comprl~ing lhe steps of (a) ple~ g ac~ntli~l~te llli2~ e of nucleic acids, (b) contacting the c~n~ te lllixLulc of nucleic acids with said cytokine, (c) partitioning between members of said~ r~n~ te mixture on the basis of affinity to said cytokine, and (d) amplifying the selected molecules to yield a 2 5 ll~i~lulc; of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to said cytokine.
More specifically, the present invention includes the RNA ligands to IFN-g,.mm~
IL~, IL-10, and TNF~ identified according to the above-described method, including those ligands shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73, 79-185; 189-205;
209-255). Also included are RNA ligands to IFN-g~mm~ I]A, IL-10, and TNF~ that are sl~st~nti~l1y homologous to any of the given ligands and that have substantially the sarne CA 02223003 l997-l2-Ol W O 96/40717 PCT~US96/09537 ability to bind IFN-~mm~ IL-4, IL-10, and TNFa and inhibit the function of IFN-g~mm~, IL-4, IL-10, and TNFa. Further included in this invention are nucleic acid ligands to IFN-g~mm~ IL-4, IL-10, and TNFa that have subst~nti~lly the same structural form as the ligands presented herein and that have substantially the same ability to bind IFN-g~mm~ IL-4, IL-10, and TNFa and inhibit the function of IFN- ~mm~ IL-4, IL-10, and TNFa.
The present invention also includes modified nucleotide sequences based on the nucleic acid ligands identified herein and ll~ixlules of the same.

n~LT~TJF',n nF~Cpc~PTION OF TRli' Tl~ TION
This application describes high-affinity nucleic acid ligands to cytokines identified through the method known as SELEX. SELEX is described in U.S. Patent ApplicationSerial No. 07/536,428, entitled Syst~m~tic Evolution of T ig,qn-l~ by EXponential Enrichment, now abandoned, U.S. Patent Application Serial No. 07/714,131, filed June 10, 1991, entitled Nucleic Acid T ig~nflc, United States Patent Application Serial No.
07/931,473, filed August 17, 1992, entitled Nucleic Acid T i~n-l~, now United States PatentNo. 5,270,163, (see also PCT/US91/04078). These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
In its most basic form, the SELEX process may be defined by the following series2 o of steps:
1) A ç~ntli~l~te llliXLul~ of nucleic acids of differing sequence is prepared. The c~n(lirl~te nlixLule generally includes regions of fixed sequences (i.e., each of the members of the c~n(~ te lllixLulc contains the same sequences in the same location) and regions of randomi~d sequences. The fixed sequence regions are selected either: (a) to assist in the 2 5 amplification steps described below, (b) to mimic a sequence known to bind to the target, or (c) to ~nh~n~e the con~ ~ntration of a given structural arrangement of the nucleic acids in the ç~n~ te mixture. The r~nrlomi7locl sequences can be totally randomi~d (i.e., the probability of finding a base at any position being one in four) or only partially r~n-lomi7P~l (e.g., the probability of finding a base at any location can be selecte-l at any 3 o level between 0 and 100 percent).

W O 96/40717 PCT~US96/09537 2) The c~n~ te llliX~ is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circl-m~t~nres, the interaction b~Lw~ell the target and the nucleic acids of the candidate mixture can be con~id~ored as forming nucleic acid-larget pairs between the target and those nucleic acids having the strongest affinity for the target.
3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) co,le~ollding to the highest affinity nucleic acids exist in the c~n~ t~ nli~lule, it is generally desirable to 10 set the partitioning criteria so that a cignific~nt amount of the nucleic acids in the c~n~ te lllix~ule (approximately 5-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new ç~nr1i~te mixture that is enriched in nucleic acids having a relatively higher affinity for the target.

5) By ,~e~Lllg the partitioning and amplifying steps above, the newly formed c~ntli~l~te llli~ C contains fewer and fewer weakly binding sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a c~n~ te mixture co..l;~ g one or a smallnumber of unique nucleic acids replP,sf~ ;..g those nucleic acids from the original 2 o candidate ",ixLu,e having the highest affinity to the target molecule.
The SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate l~ix~ule, and methods for amplifying partitioned nucleic acids to generate enriched candidate llliXLul~;. The SELEX Patent Applications also 2 5 describe ligands obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.
The nucleic acid ligands described herein can be complexed with a lipophilic compound (e.g., cholesterol) or ~tt~ched to or en- ~ps~ ted in a complex comprised of lipophilic components (e.g., a liposome). The complexed nucleic acid ligands can enhance 3 o the cellular uptake of the nucleic acid ligands by a cell for delivery of the nucleic acid ligands to an intracellular target. U.S. Patent Application No. 08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes," which is incorporated in its entirety herein, describes a method for plGp~;llg a thGld~GuLic or diagnostic complex compri.ee~l of a nucleic acid ligand and a lipophilic compound or a non-imml-nogenic, high molecular weight compound.
The methods described herein and the nucleic acid ligands identified by such methods are useful for both therapeutic and diagnostic purposes. Therapeutic uses include the tre~tnnent or ~ /elllion of r1iee~ee~e or medical con~litione in human patients.
Diagnostic lltili7~ti~n may include both in vivo or in vitro diagnostic applications. The SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications. SELEX
ntifies nucleic acid ligands that are able to bind targets with high affinity and with surprising specificity. These characteristics are, of course, the desired plopGllies one skilled in the art would seek in a diagnostic ligand.
The nucleic acid ligands of the present invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the art. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes.
Those skilled in the art would also be able to adapt any nucleic acid ligand by procedures 2 o known in the art to incorporate a labeling tag in order to track the presence of such ligand.
Such a tag could be used in a number of diagnostic procedures. The nucleic acid ligands to cytokines described herein may specifically be used for identification of the cytokine ploteins.
SELEX provides high affinity ligands of a target molecule. This rel)lesGlll~ a 2 5 singular achievement that is unprece~lçntec~ in the field of nucleic acids research. The present invention applies the SELEX procedure to the specific target. In the Example section below, the eA~Ihllental parameters used to isolate and identify the nucleic acid ligands to cytokines are described.
In order to produce nucleic acids desirable for use as a pharmaceutical, it is 3 o 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 W O 96/40717 PCT~US9G~5~7 effect; (3) be as stable as possible, and (4) be a specific ligand to the chosen target. In most situations, it is ~l~f~lled that the nucleic acid ligand have the highest possible affinity to the target.
In co-pending and commonly ~ nPd U.S. Patent Application Serial No.
5 07/964,624, filed October 21, 1992 ('624), methods are described for obtaining improved nucleic acid ligands after SELEX has been ~,.r~llllcd. The '624 application, entitled Methods of Producing Nucleic Acid r i~n-1~, is specifically incoll,ul~d herein by reference.
This invention includes the SELEX process for identification of nucleic acid 1 0 ligands of cytokin~e Cytokines are a diverse group of small proteins that mP~ te cell ~ign~lin~oll~ ication. Cytokines include jmm~lnPI hematopoietins (e.g.,EPO, GM-CSF, G-CSF, LIF, OSM, CNTF, GH, PRL, IL-2, IL-3, Il,-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12), i~lL~,rel~ns (e.g.,IFNa, IFN~, IFN-gamma), TNF-related molecules (e.g.,TNFa, IFN~, gp39 (CD40-L), CD27-L, CD30-L,NGF), and chemokines (e.g, PF4, PBP, groa, MIG, ENA-78, MIPla, MIP1~, MCP-l, I-309, HlC14, C10, RANTES, IL-8, MIP-l). In one embodiment, cytokines are derived from T-lyrnphocytes.
In the present invention, SELEX ~ ent~ were performed in order to identify RNA with specific high affinity for the cytokines IFN-g~mm~ IL-4, IL-10, hTNF~, and RANTES from degenerate libraries cont~ining 30 or 40 random positions (40N for 20 IFN-g~mm~ IL-4,IL-10andRANTES;30NforhTNFa)(Tables 1,5,9, ll,and 16).
This invention includes the specific RNA ligands to IFN-g~mm~ IL-4, IL-10, and TNFa shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73; 79- 185; 189-205; 209-255), identifie~l by the methods described in Fx~mples 1, 3, ~, 7, and 12. This invention further includes RNA ligands to IFN-g~mm~ IL-4, IL-10, and TNF~ which inhibit the function of 25 IFN-gzlmm~ IL-4, IL-10, and TNFa. This invention further includes DNA ligands to RANTES which inhibit the function of RANTES. The scope of the ligands covered bythis invention extends to all nucleic acid ligands of IFN-g~n ml~ IL-4, IL-10, TNF a, and RANTES modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are sllkst~nti~lly 3 o homologous to the ligands shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73;
79-185; 189-205; 209-255). By substantially homologous it is meant a degree of primary W O 96/40717 PCT/U~ 5537 sequence homology in excess of 70%, most preferably in excess of 80%. A review of the sequence homologies ofthe ligands of IFN-p~mm~, IL-4, IL-10, and TNFa shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73; 79-185; 189-205; 209-255) shows that sequences with little or no l~hll~y homology may have subst~nti~lly the same ability to 5 bind IFN-P;~mm~, IL-4, IL-10, and TNFa. For these reasons, this invention also includes nucleic acid ligands that have ~U~ ly the same structure and ability to bindIFN-~mm~ IL-4, IL-10, and TNFa as the nucleic acid ligands shown in Tables 3, 4, 7, 8, 10, and 12 (SEQ ID NOS:7-73; 79-185; 189-205; 209-255). SubsL~Iially the same ability to bind IFN-g~mm~ IL-4, IL-10, and TNFa means that the affinity is within one or two 0 orders of m~nitn-lç of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to ~letermine whether a given sequence --substantially homologous to those specifically described herein -- has substantially the same ability to bind IFN-g7mm~, IL-4, IL-10, and TNFa.
This invention also includes the ligands as described above, wherein certain 15 chemical modifications are made in order to increase the in vivo stability of the ligand or to çnh, nçe or mç~ te the delivery of the ligand. Examples of such modifications include chemical ~ul ~I;I--I;onc 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.i~n-l~ Co..~ in~ Modified 2 0 Nucleotides which is specifically ..,co,~o.dLed herein by reference. Other modifications are known to one of o~ ~y skill in the art. Such modifications may be made post-SELEX (modification of previously itl~ntified nnmotlified ligands) or by incorporation into the SELEX process.
As described above, because of their ability to selectively bind IFN-g;~ nm~, IL~, 25 IL-10, hTNFa, and RANTES, the nucleic acid ligands to IFN-p;~mm~ IL~, IL-10,TNF~, and RANTES described herein are useful as ph~rm~ceuti~ This invention, therefore, also includes a method of inhibiting cytokine function by ~lmini~tration of a nucleic acid ligand capable of binding to a cytokine.
Thc.~c.lLic compositions ofthe nucleic acid ligands may be ~-lmini~tered 3 o parenterally by injection, although other effective ~-1minictration forms, such as il~L~ icular injection, inh~l~nt mists, orally active formulations, transdermal iontophoresis or suppositories, are also envisioned. One ~l~r~ d carrier is physiological saline solution, but it is contemplated that other ph~rm~reutically acceptable carriers may also be used. In one ~l-r~ d embodiment, it is envisioned that the carrier and the ligand con~liLule a physiologically-compatible, slow release formulation. The primary solvent in 5 such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other ph~rm~-~ologically-acceptable excipients for modifying or m~ the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the form~ tion. Similarly, the carrier may contain still other ph~rm~t ologically-acceptable excipients for modifying or IIIAilll;1;llillg the stability, rate of 10 dissolution, release, or absorption of the ligand. Such excipients are those substances usually and customarily employed to formlll~te dosages for parental ~ mini~tration in either unit dose or multi-dose form.
Once the thc.d~c.llic composition has been formlll~t~-l it may be stored in sterile vials as a solution, ~u~ell~ion, gel, emulsion, solid, or dehyclrated or lyophili7~1 powder.
5 Such formulations may be stored either in a ready to use forrn or requiring reconstitution immediately prior to ~lmini~tration The manner of ~tlmini~t~ring formulations coll~ nucleic acid ligands for systemic delivery may be via subcutaneous, intr~mll~clll~r, intravenous, hllldnasal or vaginal or rectal suppository The following Examples are provided to explain and illustrate the present 2 o invention and are not int~ntle~ to be limitin~ of the invention.

EXAMPLE 1. EXPERIMENTAL PROCEDURES FOR 2 '-NH2 AND 2 '-F-MODIFIED LIGANDS TO IFN-GAMMA
This example provides general procedures followed ~md incorporated in Example 2 2 5 for the evolution of nucleic acid ligands to IFN-g~mm~
A. Oligonucleotides 2'F modified CTP and UTP were ~ ed according to the method of Pieken et al., - 1991. 2'NH2 modified CTP and UTP were p~ ~ed according to the method of McGee et al., U.s. Patent Application No. 08/264,029, filed June 22, 1994, which is incol~oldl~d 3 o herein by reference (see also McGee et al. 1995). DNA oligonucleotides were synth.o~i7~fl by Operon Technologies (~l~me~ CA).

W O 96/40717 PCTrUS96/09537 B.SELEX
The SELEX procedure has been described in detail in U.S. Patent No. 5,270,163 (see also Tuerk and Gold, 1990; Gold et al., 1993). Three SELEX procedures were p.. r(J....ed to evolve high affinity ligands to IFN-g~mm~ Each SELEX procedure utilized 5 RNA pools cu--L~;..in~ pyrimic~in~s modified at the 2' position as follows, 1) 2'F-CTP and 2'F-UTP referred to as 2'F, 2) 2'F-CTP and 2'NH2-UTP referred to as 2'F/NH2, and 3) 2'NH2-CTP and 2'NH2-UTP referred to as 2'NH2. For each SELEX, the DNA template 40N7 was desiPned to contain 40 random nucleotides, flanked by 5' and 3' regions of fixed sequence (Table l; SEQ ID NO:l). The fixed regions include DNA primer ~nne~linp sites 1 0 for PCR and cDNA synthesis as well as the consensus T7 promoter region to allow in vitro trsln~.rirtion.
Single-stranded DNA primers and templates were synth~ci7~od and amplified into double-str~n~1ed transcribable templates by PCR. PlGl)~dLion of the initial pool of RNA
molecules involved PCR amplification of 1000 pmoles of single-stranded template (Table 5 1; SEQ ID NO:l) and 2500 pmoles of both the 5'- (SP7; SEQ ID NO:2) and 3'- (3P7; SEQ
ID NO:3) prim~rs These were incllb~tecl in a reaction mixture co..~;..;..~g 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 0.5 mM of each dATP, dCTP, dGTP, and dTTP. Taq DNA Polymerase (Perkin-Elmer, Foster City CA) at 0.1 U/~l was added and the reaction incubated at 97~C for 3 min to denature the template and primers. Following the initial denaturing step, the reaction was cycled 10 times at 93~C for 30 sec, 53OC for 30 sec, and 72~C for 1 min to denature, anneal, and extend, respectively, the primers and template. To get an ~c~lr~te concentration of double-stranded PCR product for the initial round of SELEX, the PCR product was purified using QIAquick-spin PCR purification columns(QIAGEN Inc., Chatsworth CA) as specified by the m~nllf~ rer.
2 5 For in vitro transcription using modified nucleotides 200 pmoles (final co~entration of 1 ,~M) of double-stranded DNA temrl~te was incubated in a reaction lwecont~ining 40 mM Tris-Cl (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM
sperrnidine, 0.002% Triton X-100. 4% PEG 8000,0.511M ~-32P-ATP,S Ut~lT7 RN A
Polymerase (Davanloo et al., 1984), and conce~ ions of other nucleotides as follows, I ) 3 o for the 2'F SELEX: I mM ATP and GTP, 3 m M 2'F-CTP and 2'F-UTP, 2) for the 2'FtNH2 SELEX: 1 mM ATP,GTP, and 2'NH2-UTP and 3 m M 2'F-CTP, and 3) for the 2rNH2 SELEX: 1 rnM ATP, GTP, 2'NH2-CTP, and 2'NH2-UTP. These in~ h~tions were performed in a 37~C in~llb~tor for between 6 hrs and overnight. Typically the RNA was - purified by gel purification and elution. To expedite the process, for rounds 1 1, 12, and 14-17 the RNA was purified using Bio-Spin 6 chromatograplly colurnns (Bio-Rad 5 Laboratories, Hercules CA) according to m~nllf~rt-lrer's specifications. To reduce background, the RNA was pre-filtered prior to all rounds of SELEX except rounds 1, 2, 4, 6, 14, and 16. The pre-filtration step involved bringing the RNA up to 200 ~1 in phosphate buffered saline (PBS), modified to contain lrnM Mg2+ ions, (138 rnM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.1 rnM KH2PO4 lmM MgCl2, pH 7.4), (rnPBS), and passing this RNA10 solution through three filter discs (0.45 ~m, nitrocellulose/ cellulose acetate, Millipore Corporation, Bedford MA) pre-wetted with rnPBS.
For initial binding, 1000 pmoles of RNA were incubated with human IFN-gamma protein in binding buffer, (rnPBS plus 0.01% hurnan serum albumin (HSA)), for 5-10 min at 37~C to allow binding to occur. Human recombinant IFN-gamma used in this SELEX
15 procedure was purchased from two di~ sources. The first three rounds of both the 2'F
and 2'F/NH2SELEX were performed with protein obtained from Upstate Biotechnology, Lake Placid NY. The subsequent rounds of these two SELEX procedures as well as the entire 2'NH2 SELEX were performed with protein obtained from Genzyme Inc., Cambridge MA. For each round of SELEX the concentration of RNA and protein was 2 o carefully chosen to provide ol,Li.llu,ll stringency. Increased stringency was obtained during rounds 8-13 of SELEX by adding NaCI to the binding buffer to bring the final chloride ion concentration up to 250 mM. Preliminary experiiments had shown thatIFN-gamma had a t~n-l~n~y to ag~leg~le at high protein concentrations. To prevent the evolution of RNA species having an affinity for this aggregated IFN-g~nnm~, beginning 2 5 wiith round 4 of SELEX and for all subsequent rounds of the SELEX procedure, the binding mix was centrifuged at 16,000 x g for 3 min in an eppendorf centrifuge before nitrocellulose filter partitioning. IFN-gamma/RNA complexes were separated from unbound RNA by nitrocellulose filter partitioning described below.
For nitrocellulose partitioning, the 2'F and 2'F/NH2 SE LEX procedures used 0.2 "m 3 o pore size pure nitrocellulose filters (Scleicher & Schuell, Keene NH) for the first two rounds of SELEX. All subsequent rounds of these two SELEX procedures and the entire W O 96/40717 PCT~US96/09537 2'NH2 SELEX were performed with 0.45 ~m pore size nitrocellulose/cellulose acetate mixed matrix filters (Millipore Corporation, Bedford MA). Filter discs were placed into a vacuum manifold and wetted with 5 ml of mPBS buffer. The IFN-gamma/RNA binding mix was aspirated through the filter discs which were immediately washed with 5 ml of mPBS buffer. To further increase stringency and reduce background for rounds 8-13, and 15, this washing step was modified to include washing ofthe filter discs with 15 ml 0.5 M
urea followed by 20 ml mPBS buffer. Bound RNA was isolated from filters by extraction in a solution of 400 m 1 phenol (equilibrated in Tris-Cl, pH 8.0)/300 m 1 7 M urea (freshly pl'~.,d). The filters were bathed in the phenoVurea solution at room te~ .a~ for 30 0 min and at 95~C for 2 min. The RNA was phenol/chloroform extracted and ethanol p~ Led with 20 mg tRNA.
The RNA was reverse transcribed into cDNA by addition of 50 pmoles DNA
primer, 0.4 mM each of dNTPs, and 1 U/~l AMV reverse transcriptase (AMV RT) (Life Sciences, Inc., St. Petersburg FL) in buffer co~ 50 mM Tris-Cl (pH 8.3), 60 mM
NaCl, 6 mM Mg(OAc)2, 10 mM DTT. The reaction was incubated at 37~C for 30 min then 48~C for 30 min then 70~C for 10 min, to ensure the melting of secondary structure present in the isolated RNA.
To begin a new round of SELEX, the cDNA was PCR amplified by addition of 250 pmoles of both the 5' (5P7; SEQ ID NO:2) and 3' (3P7; SEQ ID NO:3) primer in reaction 2 o conditions identical to those detailed above. The number of cycles of PCR required to amplify the cDNA was carefully calculated for each round of SELEX so that 250 pmoles double-stranded DNA template would be used to initiate the next round of SELEX.

C. Equilibrium D;,so~;~lion Constants (Kds) 2 5 The dete~ " ,i"~tion of equilibrium dissociation con~ (Kds) for RNA pools was made subsequent to rounds 5, 8, 12, and 17 to monitor the progress of each SELEX . The Kds of RNA pools for mouse IFN-gamma (Genzyme Inc., Cambridge MA) were also det~rrnin.od after rounds 8 and 17. Kds were deterrnin~d for individual ligands after cloning and seqll~onrin~ of RNA pools and truncations (described below). Nitrocellulose 3 o filter binding was used to determine Kds as follows: filter discs were placed into a vacuum manifold and wetted with S ml of mPBS buffer. '~P-labeled-RNA was inc~lb~ttod W O 96/40717 PCT~US96/09537 with serial dilutions of IFN-garnma in binding buffer for 5-l0 min at 37~C to allow binding to occur. Binding mixes were cc;~ iruged as described above to remove aggregates, ~epir~te~l through the filter discs, and then imrned~iately washed with 5 ml mPBS buffer. The filter discs were dried and counted in a liquid.scintill~tion counter 5 (Be~m~nn Instrllm~nte, Palo Alto CA). Equilibrium dissociation co~ were ~le~ ....;-.ecl by least square fitting ofthe data points using t_e KaleidagraphrM graphics program (Synergy Software, Reading PA). Many ligands and evolved RNA pools yieldbiphasic binding curves. Biphasic binding can be described as the binding of two affinity species that are not in eqllilibrillm Biphasic binding c~ were calculated according 10 to standard procedures. Kds were d~ by least square fitting of the data points using the Kalei~ phTM graphics program.

D. Cloning and Sequencing After the l 7th round of SELEX, RNA molecules were reverse transcribed to cDNA
5 and made double-stranded by PCR amplification with primers co..l~ recognition sites for the restriction en~lQnllcleases Hind III (Table l; 5' primer 5P7H; SEQ ID NO:4) and Bam HI (Table l; 3' primer 3P7B; SEQ ID NO:5). Using these restriction sites the DNA
sequences were inserted directionally into the pUCl9 vector. These recombinant plasmids were traneformed into Epicurian coli JMl09 competent cells (Stratagene, La Jolla CA).
2 o Plasmid DNA was ~ a,ed with the PERFECTprepTM plasmid DNA kit (5 prime--->3 prime, Boulder CO). Plasmid clones were sequenced using a PCR sequencing protocol (Adams et al., l99l) using PCR sequencing primer pUCl9F30 (SEQ ID NO:6).

E. Ligand T.. _ -n Boundary ~ye~ nte were carried out to determine the minim~l sequence necessary for high affinity binding of the RNA ligands to IFN-gamma using end-labeled RNA. Prior to end-labeling, RNA transcribed with T7 RNA polymerase was gel purified by W shadowing. The 5'-end of 20 pmoles of each RNA w~s dephosphorylated in a reaction mixture coll~ g 20 mM Tris-Cl (pH 8.0), l0 mM: MgCl2 and 0.l U/~l shrimp 3 o ~lk~lin.o phosphatase (SAP), (United States Biochemical, Cleveland OH) by incubating for 30 min at 37~C. Alkaline phosphatase activity was destroyed by incubating for 30 min at W O 96/40717 PCT~US96/09537 70~C. RNA was subsequently 5'-end labeled in a reaction mixture co.~ .g 50 mM
Tris-Cl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 0.1 mM spermidine, 0.75 m M g -32P-ATP and 1 U/~l T4 polynucleotide kinase (New Fngl~n-l Biolabs, Beverly MA) by incubating for 30 min at 37~C.
3'-end-labeling of 20 pmoles of each RNA was performed in a reaction mixture COI.~ il.g 50 mM Tris-Cl (pH 7.8), 10 mM MgCl2, 10 mM b -mercaptoethanol, 1 mM
ATP, 0.9 m M (S'-32P)pCp and 1 U/~l T4 RNA ligase (New Fngl~n-l Biolabs, Beverly- MA) by inr~lb~tin~ for 18 hrs at 4~C. 5'- and 3'- end-labeled RNAs were gel band purified on a 12%, 8M urea, polyacrylamide gel. After partial ~lk~line hydrolysis of the end-labeled RNA by addition of Na2CO3 to a final concentr~tion of 50 mM and incubation in a boiling water bath for 3 min, radiolabeled RNA ligands were incubated with IFN-gamma at three di~clclll protein concentrations, 1) 5-fold below the ~ xhllate Kd, 2) at the a~ro~il--~te Kd, and 3) 5-fold above the a~J~lvxi~ lc Kd. Protein-bound RNA
was scpaldled by nitrocellulose partitioning. RNA truncates were analyzed on a high-resolution d~n~tllring 12% polyacrylamide gel. To orient the sequences, a ladder of radioactively labeled ligands tt?rmin~tin~ with G-residues was generated by RNase T1 digestion of end-labeled RNA. The Tl digest was carried out in a reaction mixture co..l~il.i-.g 7 M urea, 20 mM sodium citrate (pH 5.0), 1 mM EDTA and S units RNase Tl (Boehringer ~nnh~im, Tn~ n~rolis IN) by inr~lb~tin~ for S min at 50~C.
2 0 Complem~nt~ry single-stranded DNA oligonucleotides co- ~ g the sequence of the T7 promoter (5'-TAATACGACTCACTATAG-3'; fr~gment of SEQ ID NO:2) and the sequence of the trllnr~tr~1 ligand were ~nnP~lerl to form a double-stranded template for transcription of each trunc~ted ligand.

2 5 F. Receptor Binding Competitions Human lung carcinoma cells (A549; ATCC) were plated in 2~well plates at a density of S X 105 cells/well in RPMI 1640 plus 10% fetal bovine serum (FBS) andinrub~t~cl overnight or until confluent. The cells were washed 3 times with PBS. Growth media was replaced with 200 ,~1 RPMI 1640 plus 0.2% human serum albumin/0.02%
3 o sodium azidel20 mM Hepes, pH 7.4 together with increasing amounts (20 pg/ml-l 00 ng/ml) of '2sI-IFN-gamma (New F.ngl~ntl Nuclear) with or without an excess (200 fold) of W O 96/40717 PCTrUS96/09537 unlabeled IFN-g~mm~ Tn~llh~ti~-ns were carried out at 4~C with ~h~king for 2 hrs. The cells were washed 2 times with cold PBS to remove free IFN and detached with 0.5%
SDS. Cell-associated '2sI-IFN-gamma was tletermined by me~Cllrin~ the radioactivity of the detached cells in a gamma counter. The data was corrected for nonspecific binding 5 and the affinity of l25I-IFN-garnma was ~l~t~rrnined by Scatchard analysis of the binding data. Scdlch~d analysis suggests that there are high-affinity binding sites (Kd = 20pM) and low-affinity binding sites (Kd = 0.5nM). For competition with oligonucleotide, the cells were inlmb~te~l for 2 hr at 4~C as above with 30 pM ~25I-IFN-garmna and increasing concentrations (1.01-500 nM) of competitor oligonucleotide. Cell-associated 10 l25I-IFN-gamma was clet~rminPd as above.

EXAMPLE 2. 2 -NH2 AND 2'-F-MODIFIED RNA LIGANDS TO IFN-GAMMA
A. SELEX
Three libraries of RNAs modified at the 2' position of pyrimitlin~s, 1 ) 2'F
15 incorporating 2'F-CTP and 2'F-UTP, 2) 2'FtNH2 incorporating 2'F-CTP and 2~H2-UTP
and 3) 2~Hz incol~oldLing 2'NH2-CTP and 2~Hz-UTP were used in cimlllt~neous SELEX
protocols to generate a diverse set of high-affinity modified RNA ligands to human IFN-g~mm~ Each ofthese libraries contained between 10~3-10~4 molecules with a variable region of 40 nucleotides The template and primers used for the SELEX and the 2 0 conditions of the SELEX, as described in Example 1, are sumrnarized in Tables 1 and 2, respectively.

B. RNA Sequences and D ~OL ";- ' Constants The random modified RNA pools bound human IFN-gamma with apploxi.l.ate 2 5 Kds of greater than 0.7 ~M. After 17 rounds of SELEX, the approximate Kds of the evolving pools had improved to, 1) 70 nM for the 2'F SELEX, 2) 115 nM for the 2'F/NH2 SELEX, and 3) 20 nM for the 2~H2 SELEX. For mouse IFN-gRmm~ the approximate Kds of the RNA pools after 17 rounds of SELEX were 1) 410 nM for the 2'F SELEX, 2) 175 nM for the 2'F/NH2 SELEX, and 3) 85 nM for the 2~H2 SELEX. These Kds did not3 o shift further in subsequent rounds.

In order to determine to what extent the evolving pool was still random, PCR
product from the final round of SELEX was sequenced as detailed above and found to be non-random. RNA from the l 7th round was reverse transcribed, amplified and cloned.
The sequences of 32 of the 2'F, 40 of the 2'NH2, and l 1 of the 2'F/NH2 individual clones 5 were ~l~termin~cl (Table 3, SEQ ID NOS:7-65). The sequences were analyzed for conserved se~luences and aligned by this criterion (Table 3). The 2'F sequences fell in to 2 groups with 9 orphan sequences. Group l 2'F RNAs were the most abundant, representing 18 of 32 sequences, while group 2 2'F RNAs ~ es~ ed 5 of 32 sequences. The 2'NH2sequences fell into 2 groups with 25 of 40 2'NH2 RNAs in group l and l 5 of 40 2'NH2 10 RNAs in group 2. The 2'F/NH2 sequences were of a single group.
The Kds of individual RNAs within each group were det~rmin~cl by nitrocelulose filter binding as described above. The Kds were llet~rmin~l using either a monophasic or biphasic least squares fit of the data.
Minimal sequence requirements for high-affinity binding of the best clones were 15 determined by 5' and 3' boundary expenm~nt~ as described. The truncated RNAs were tr~n~- rihed from double-stranded templates cO.~t~ g the T7 promoter and the trlln-~tç
sequence. For those snrcç~ l transcriptions, the Kd of the truncated ligand was let~rrnined. The sequence of the truncated ligands and their Kds, both for full-length and for the truncate (if ~l~t~rmine~l) are shown in Table 4 (SEQ ID NOS:66-73).
C. Receptor Competition Both full-length 2'NH2 (2'NH2 ~ dom, 2 'NH2 ,, 2 'NH2 30~ and 2 'F (2 'F random, 2'F-1, and 2'F-28) oligonucleotides were tested for their ability to inhibit receptor binding. This competition was targeted primarily to the high-affinity binding component using a 2 5 concentration of l25I-IFN-gamma of 30pM. At this concentration, neither the 2 'NH2 nor the 2'F random oligos showed inhibition, while varying degrees of inhibition were seen with the 4 clones tested. The 2'NH2 ligand #30 (SEQ ID NO:72) was the best inhibitor and showed 50% inhibition at l0 nM.

W O 96/40717 PCTAUS96/09~37 EXAMPLE 3. EXPERIMENTAL PROCEDURES FOR ~'-N H2 AND

This Exarnple provides general procedures followed and incorporated in Example 4 for ~he evolution of nucleic acid ligands to IL-4.

A. Oligonucleotides 2'F modified CTP and UTP were prepared according l:o the method of Pieken et al., 1991. 2'NHz modified CTP and UTP were ~l~paled according to the method of McGee et al., U.s. Patent Application No. 08/264,029, filed June 22, 1994, which is incorporated 0 herein by reference (see also McGee et al. 1995). DNA oligollucleotides were syntht-si7~cl by Operon Technologies (~l~m~ CA).

B. SELEX
The SELEX procedure h~ been described in detail in U.S. Patent No. 5,270,163 15 (see also Tuerk and Gold, 1990, Gold et al., 1993). Three SELEX procedures were olllled to evolve high affinity ligands to IL-4. Each SELE,X procedure utilized RNA
pools COI~t~ pyrimil1in-o~ modified at the 2' position ~ follows, 1) 2'F-CTP and2'F-UTP referred to as 2'F, 2) 2'F-CTP and 2~H2-UTP referred to ~ 2'F/NH2, and 3) 2 NH2-CTP and 2 NH2-UTP referred to ~ 2'NH2. For each SELEX, the DNA template 2 o 40N8 w~ clçci~n~cl to contain 40 random nucleotides, flanked by 5' and 3' regions of fixed sequence (Table 5; SEQ ID NO:74). The fixed regions includle DNA primer ~nnr~ling sites for PCR and cDNA synthesis ~ well ~ the con~- n~ T7 promoter region to allow in vitro transcription.
Single-st~nc~ DNA primers and templates were syntht?si7t?cl and amplified into 2 5 double-str~n~le~l transcribable templates by PCR. Ple~lion of the initial pool of RNA
molecules involved PCR amplification of 1000 pmoles of single-stranded template (Table S; SEQ ID NO:74) and 2500 pmoles of both the 5' (SP8; SEQ ID NO:75) and 3' (3P8;- SEQ ID NO:76) primers. These were incubated in a reaction mixture CO~ g 50 mM
KCl, 10 mM Tris-Cl (pH 8.3), 3 mM MgCl2, 0.5 mM of each dATP, dCTP, dGTP, and 3 o dTTP. Taq DNA Polymerase (Perkin-Elmer, Foster City CA) at 0.1 U/,~l was added and the reaction incubated at 97~C for 3 min to denature the template and primers. Following W O 96/40717 PCTrUS96/09537 the ir~itial clenAhlring step, the reaction was cycled 7 times at 93~C for 30 sec, 53~C for 30 sec, and 72~C for 1 min to denature, anneal, and extend, respectively, the primers and template. To get an accurate concentration of double-stranded PCR product for the initial round of SELEX, the PCR product was purified using QIAquick-spin PCR purification 5 columns (QIAGEN Inc., Chdl~w~Jllh CA) as specified by the mAnllf~rturer.
For in vitro transcription using modified nucleotides 200 pmoles (final concentration of 1 ~M) of double-strAntled DNA template was incubated in a reaction i~LulG co..~ 40 mM Tris-Cl (pH 8.0), 12 mM MgCl2, 5 mM DTT, 1 mM
spermidine, 0.002% Triton X-100, 4% PEG 8000, 0.5 ~M oc-32P 2'0H ATP, S U/~l T7 10 RNA Polymerase (Davanloo et al., 1984), and concentrations of other nucleotides as follows, 1) for the 2'F SELEX: 1 mM ATP and GTP, 3 mM 2'F-CTP and 2'F-UTP, 2) for the 2'F/NH2 SELEX: 1 mM ATP, GTP, and 2'NH2-UTP and 3 mM 2'F-CTP, and 3) for the 2'NH2 SELEX: 1 mM ATP, GTP, 2'NH2-CTP, and 2'NH2-UTP. These incubations were pGlrc"llled in a 37~C incubator for between 6 hrs and overnight. Typically the RNA
15 was purified by gel purification and elution. To G~edil~ the process for rounds 11, 12, and 14-17 the RNA was purified using Bio-Spin 6 chromatography columns (Bio-Rad Laboratories, Hercules CA) according to mAmlfArhlrer's specifications. To reducebackground, the RNA was pre-filtered prior to all rounds of SELEX except rounds 1, 2, 4, 6, 14, and 16. The pre-filtration step involved bringing the RNA up to 200 ~1 in phosphate 2 o buffered saline (PBS), modified to contain 1 mM Mg2+ ions, (138 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.1 mM KH2PO4 lmM MgCl2, pH 7.4), (mPBS), and passing this RNA
solution through three filter discs (0.45 ~rn, nitrocellulose/ cellulose acetate, Millipore Corporation, Bedford MA) pre-wetted with mPBS.
For initial binding, 1000 pmoles of RNA were in~ ub~ted with human IL-4 protein 25 in binding buffer, (mPBS plus 0.01% human serum alburnin (HSA)), for 5-10 min at 37 ~C to allow binding to occur. Human recombinant IL~ used in this SELEX procedurewas purchased from R & D Systems, Mirmeapolis MN. For each round of SELEX the co~ lion of RNA and protein was carefully chosen to provide optimurn stringency.Prelimin~ Ap~,;l"ents had shown that IL~ had a tendency to aggregate at high protein 3 o conce~ dlions. To prevent the evolution of RNA species having an affinity for this aggregated IL-4, beginning with round 4 of SELEX and for all subsequent rounds of the W O 96/40717 PCT/U',G~'~9S37 SELEX procedure, the binding mix was centrifuged at 16,000 X g for 3 min in an eppendorf centrifuge before nitrocellulose filter partitioning. IL-4/ RNA complexes were separated from unbound RNA by nitrocellulose filter partitioming described below.
For nitrocellulose partitioning, the 2'F and 2'F/NH2 SELEX procedures used 0.2 ,~m 5 pore size pure nitrocellulose filters (Scleicher & Schuell, Keene NH) for the first two rounds of SELEX. All subsequent rounds of these two SELEX procedures and the entire 2rNH2 SELEX were performed with 0.45 ,~m pore size nitrocellulose/cellulose acetate mixed matrix filters (Millipore Co~ dlion, Bedford MA). Filter discs were placed into a vacuum manifold and wetted with 5 ml of mPBS buffer. The IL-4/RNA binding mix was 10 aspirated through the filter discs which were imm~ t~1y washed with 5 ml of mPBS
buffer. To further increase stringency and reduce background for rounds 8-13, and 15, this washing step was modified to include washing ofthe filter discs with 15 ml 0.5 M urea followed by 20 ml mPBS buffer. Bound RNA was isolated from filters by extraction in a solution of 400 ~1 phenol (equilibrated in Tris-Cl, pH 8.0)/ 300 ~1 7 M urea (freshly 15 prepared). The filters were bathed in the phenol/urea solution at room te.l.pe.~ for 30 min and at 95~C for 2 min. The RNA was phenol/chlolorol."l extracted and ethanolprecipitated with 20 ~g tRNA.
The RNA was reverse transcribed into cDNA by addition of 50 pmoles DNA
primer, 0.4 mM each of dNTPs, and 1 U/~l AMV reverse transcriptase (AMV RT) (Lif2 o Sciences, Inc., St. Petersburg FL) in buffer co. .~ .;, .g 50 mM Tris-Cl (pH 8.3), 60 mM
NaCl, 6 mM Mg(OAc)2, 10 mM DTT. The reaction was inrllb~te-l at 37~C for 30 min then 48~C for 30 min then 70~C for 10 min, to ensure the melting of secondary structure present in the isolated RNA.
To begin a new round of SELEX, the cDNA was PCR amplified by addition of 250 2 s pmoles of both the 5' (SP8; SEQ ID NO:75) and 3' (3P8; SEQ ID NO:76) primer in reaction conditions identical to those detailed above. The number of cycles of PCR
required to amplify the cDNA was carefully calculated for each round of SELEX so that 250 pmoles double-stranded DNA template would be used to initiate the next round of SELEX.

W O 96/40717 PCT~US96/09537 C. Equilibrium D;i.so~;~licConstants (Kds) The ~ tion of equilibrium dissociation co~ (Kds) for RNA pools was made subsequent to rounds 5, 8, 12, and 17 to monitor the progress of each SELEX. The Kds of RNA pools for mouse IL-4 (R & D Systems, Mirmeapolis MN) were also 5 ~let~rmined after round 8. Kds were .1~ i..ecl for individual ligands after cloning and sequencing of RNA pools and truncations (described below). Nitrocellulose filter binding was used to ~letermine Kds as follows: filter discs were placed into a vacuum manifold and wetted with 5 ml of mPBS buffer. 32P-labeled-RNA was incubated with serial dilutions of IL-4 in binding buffer for 5-10 min at 37~C to allow binding to occur.
0 Binding mixes were centrifuged as described above to remove aggregates, aspirated through the filter discs, and then immefli~tely washed with 5 ml mPBS buffer. The filter discs were dried and counted in a liquid scintill~tion counter (Becl~ ol- Instrnment~, Palo Alto CA). Equilibrium dissociation constants were clett~rminecl by least square fitting of the dat~ points using the KaleidagraphrM graphics program (Synergy Software, Reading 15 PA). Many ligands and evolved RNA pools yield biphasic binding curves. Biphasic binding can be described as the binding of two affinity species that are not in equilibrium.
Biphasic binding CU~ were calculated according to standard procedures. Kds were determined by least square fitting of the data points using the KaleidagraphTM graphics program.
D. Cloning and Sequencing After the 1 7th round of SELEX, RNA molecules were reverse transcribed to cDNA
and made double-stranded by PCR amplification with primers co..l ~ recognition sites for the restriction endonucleases Hind III (Table 5; 5' primer 5P8H; SEQ ID NO:77) and 25 Bam HI (Table 5; 3' primer 3P8B; SEQ ID NO:78). Using these restriction sites the DNA
sequences were inserted directionally into the pUC l 9 vector. These recombinant plasmids were transformed into Epicurian coli JM109 co~ .lt cells (Stratagene, La Jolla CA).
Plasmid DNA was ~ lep~ed with the PERFECTpreprM plasmid DNA kit (5 prime--->3 prime, Boulder CO). Plasmid clones were sequenced using a PCR seqllencing protocol 3 o (Adams et al., 1991 ) using PCR seq~l~n~in~ primer pUC I 9F30 (SEQ ID NO:6).

E.Ligand Truncation Boundary experim~nt~ were carried out to det~rrnin~ the minim~l sequence necessz" ~ for high affinity binding of the RNA ligands to IL-4 using end-labeled RNA.
Prior to end-labeling, RNA transcribed with T7 RNA polymerase was gel purified by UV
5 shadowing. The 5'-end of 20 pmoles of each RNA was dephosphorylated in a reaction lllixlule co.ll;1;..;.,g 20 mM Tris-Cl (pH 8.0), l0 mM MgC12 and 0.l U/~l shrimp ~lk~line phosI)h~t~e (SAP), (United States Biochemical, Cleveland OH) by in~mbating for 30 min at 37~C. ~lk~lin~ phosph~t~e activity was destroyed by incubating for 30 min at 70~C.
RNA was subsequently 5'-end labeled in a reaction llliX~ cc.. ~ g 50 mM Tris-Cl (pH
10 7.5), l0 mM MgCl2, 5 mM DTT, 0.l mM EDTA, 0.l mM sperrni-lin~, 0.75 m M g -32P-ATP and 1 U/,ul T4 polynucleotide kinase (New Fngl~nrl Biolabs, Beverly MA) by incubating for-30 min at 37~C.
3'-end-labeling of 20 pmoles of each RNA was pe,r",ned in a reaction mixlu,e co"l~ 50 mM Tris-Cl (pH 7.8), l0 mM MgCl2, l0 mM b -mercaptoethanol, l mM
5 ATP, 0.9 ,~M (5'-32P)pCp and 1 U/~l T4 RNA ligase (New F.n~l~n-l Biolabs, Beverly MA) by incubating for 18 hrs at 4~C. 5'- and 3'- end-labeled RNAs were gel band purified on a 12%, 8M urea, polyacrylarnide gel. After partial ~lk~line hydrolysis of the end-labeled RNA by addition of Na2CO3 to a final con~tontr~tion of 50 mM and incubation in a boiling water bath for 3 min, radiolabeled RNA ligands were incubated with IL-4 at three di~,~
2 o protein concentrations, l) 5-fold below the approxim~te Kd, 2) at the approximate Kd, and 3) 5-fold above the appro~im~te Kd. Protein-bound RNA was s~aled by nitrocellulose partitioning. RNA truncates were analyzed on a high-resolution d~n~hlring 12%
polyacrylamide gel. To orient the sequences, a ladder of radioactively labeled ligands t~rmin~ting with G-residues was generated by RNase Tl digestion of end-labeled RNA.
2 5 The T1 digest was carried out in a reaction ~fixlu,e co"l~;~.;.,g 7 M urea, 20 mM sodium citrate (pH 5.0), l mM EDTA and 5 units RNase Tl (Boehringer Mannheim, Tn~ n~polis IN) by incubating for 5 min at 50~C.
Compl~m~nt~ry single-stranded DNA oligonucleotides cont~inin~ the sequence of the T7 promoter (5'-TAATACGACTCACTATAG-3'; fragment of SEQ ID NO:75) and the 3 0 sequence of the l~ ed ligand were annealed to form a double-stranded template for transcription of each trnn~ ted ligand.

W O 96t40717 PCTAUS96/09537 F. Receptor Competition Human T-cell lymphoma cells (H-9; ATCC) were cultured in suspension in RPMI
1640 + 10% FCS. Cells were washed two times with PBS and rçsuspended (5.0 x 105 5 cells) in 200 ~1 media co~ in;~ RPMI 1640 + 0.02% human serum albumin/0.2% Na azide/20 mM HEPES, pH 7.4 for 2 hr at 4~C in 1.5 ml polypropylene tubes (Eppendorf, W. C..lllal~y) with various amounts of '2sI-rIL-4 in the presence or absence of a 200-fold excess of unlabeled cytokine. Following in-~lb~tion, the tubes were spun (150 x g, 5 min, 4~C) and the ~u~ ..l was aspirated. The cell pellet was resuspended in 200 ~1 10 RPMI-HSA. 100 ul aliquots were centrifuged through a cushion of an equal volume of phth~l~te oils (dibutyl/dioctyl, 1:1 v/v). The tube was rapidly frozen in dry ice/ethanol and the tip co..l~ the cell pellet w~ cut off and placed in a vial for gamma counting. The data was cc~lle~;lGd for nonspecific binding and the affinity of l25I-IL-4 was determined by Scatchard analysis. For competition with oligonucleotide, or neutralizing antibody (R &
D Systems), the cells were incubated for 2 hr at 4~ as above with 0.7 nM '25I-IL~ and increasing concentrations (0.01-500 nM) of competitor oligonucleotide. Cell-associated 5I-IL-4 was ~letPrminP(l as above.

~XAlVlP~.F 4. 2'-NH2 AND 2'-F-MODIFIED RNA LIGANDS TO IL-4 2 o A. SELEX
Three libraries of RNAs modified at the 2' position of pyrimi~1in~s, 1) 2'F
hlcol~ol~ g 2'F-CTP and 2'F-UTP, 2) 2'F/NH2 incorporating 2'F-CTP and 2rNE~2-UTPand 3) 2'NH2 incorporating 2rNH2-CTP and 2~NH2-UTP were used in simultaneous SELEX
protocols to generate a diverse set of high-affinity modified RNA ligands to human IL-4.
2 5 Each of these libraries cont~in~cl bGLv eGll 10'3-10'4 molecules with a variable region of 40 nucleotides. The tPrnpl~te and primers used for the SELEX and the conditions of the SELEX, as described in Example 3 are su~ ed in Tables S and 6, respectively.

B. RNA Sequences and Di rQ --ti2 Constants 3 o The random modified RNA pools bound human IL-4 with approximate Kds ofgreater than 20 ,~M. After 17 rounds of SELEX. the approximate Kds of the evolving W O 96/40717 PCT~US96/09537 pools had improved to, 1) 30 nM for the 2'F SELEX, and 2) 55 nM for the 2'F/NH2 SELEX. Binding curves performed on 2~H2 RNA from an earlier round had shown an approximate Kd of 100 nM, however, difficulties with background reduction in this SELEX led to an a~ l Kd after round 17 of 1 ,L.M. It was felt that despite this 5 "m~kinp" due to background, the high affinity unique sequence 2'NH2 RNAs were still in the pool after round 17. These Kds did not shift further in sulbsequent rounds. The RNA
pools after 8 rounds of SELEX did not bind mouse IL-4, while there was a significant ov~lllent in binding after 8 rounds for the human protein (data not shown).
In order to ~çt~rmine to what extent the evolving poo]l was still random, PCR
0 product from the final round of SELEX was sequenced as delailed above and found to be non-random. RNA from the 17th round was reverse transcribed, ~mplified, and cloned.
The sequences of 41 of the 2'F, 57 of the 2rNH2, and 30 of the 2'F/NH2 individual clones were ~leterrnine~l (Table 7; SEQ ID NOS:79-177). The sequences were analyzed forconserved sequences and aligned by this criterion (Table 7). The 2'F sequences fell into a 5 single group represçntin~ 29 of 41 sequences. The rem~ining 12 clones were categorized as orphans due to their lack of sequence homology with the primary group or to each other. The 2~H2 sequences fell into 2 distinct groups of sequences. Group 1 which represented 21 of 57 sequences were shown to bind to IL-4. The other group, representing 35 of 57 sequences were shown to bind to nitrocellulose filters. The presence of such a 2 0 large nurnber of nitrocellulose filter binding RNAs was not a surprise as these sequences were cloned from a pool with high background binding. These nitrocellulose binding RNAs are identified by the presence of a direct repeat of the sequence GGAGG. A single orphan 2~H2 sequence was also found. The 2'F/NH2 sequences were more heterogeneous with sequences falling into 3 groups. RNAs in group 1 and 2 bound to IL~, while the 3rd 25 group bound to nitrocellulose filters. The clones in the nitrocellulose filter binding group also contained a single or repeat of the sequence GGAGG. It should be noted that this sequence is also found in the 3'-fixed region (underlined in Table 7).
The Kds of individual RNAs within each group were ~çt-?rmint?d by nitrocelulose filter binding as described in Example 3 above. The Kds were determined using a 3 o monophasic least squares fit of the data.

W O 96/40717 PCTrUS96/09537 Minimal sequence requirements for high-affinity binding of the best clones were ~let~rmin~-l by 5' and 3' boundary exp~rim~nt~ as described in Example 3. The tr mrz~t~?cl RNAs were transcribed from double-stranded templates co--f;~ the T7 promoter andthe trlln~ ~t~ sequence. For those sl-cce~!~rul transcriptions, the Kd of the tr -nc~te~l ligand was ~ t~rmin~-1 The sequence of the l~ ri1~e~l ligands and their Kds, both for full-length and for the truncate (if ~ . " ~ ) are shown in Table 8 (SEQ ID NOS : 1 78- 185).

C. Receptor Competition Full-length 2~H2 (2~H2 random, 2'NH2-29), 2'F (2'F random, 2'F-9) and 2'F/NH2 (2'F/NH2 r~nflQm, 2'F/NH2-9 and 2'F/NH2-28) oligonucleotides were tested for their ability to inhibit receptor binding. Neither the 2'NH2, 2'F, or 2'F/NH2 random oligos showed inhibition, while varying degrees of inhibition was seen with the clones tested. At an IL-4 concentration of 0.7 nM the 2'F/NH2 ligand-9 was the best competitor for receptor binding and showed 50% inhibition at approximately 40 nM. The competition by this oligonucleotide was similar to that seen by a neutralizing antibody to IL~.

EXAMPLE 5. EXPERIMENTAL PROCEDURES FOR 2 -F MODIFI~:D

This Example provides general procedures followed and incul~oldLed in Example 6 forthe evolution of nucleic acid ligands to IL-10.

A. Materials DNA sequences were synth~si7~1 by using cyanoethyl phosphoramidite under standdrd solid phase chPmi~try. 2'-F CTP and 2'-F UTP were p~chdsed from United States Biochemic~l~. Human IL-10 was bought from either Bachem or R&D Systems.
Neutralizing anti-human IL-10 monoclonal antibody, murine IL-10 and ELISA detection kit for human IL-l 0 were purchased from R &D Systems.

B. SELEX
3 o Five nmoles of synthetic DNA template, that was purified on an 8%
polyacrylarnide gel under de.,aLuliilg conditions were amplified by four cycles of polymerase chain reaction (PCR). The PCR products were transcribed in vitro by T7 RNA polymerase (1000 U) in 1 mL reaction con~i.ctin~ of 2 mM each of ATP and GTP, 3 - mM each of 2'-F CTP and 2'-F UTP, 40 mM Tris-HCl (pH 8.0), 12 mM MgCl~, 1 mM
Sp~nni~lin~, 5 mM DTT, 0.002% Triton X-100 and 4% polyethelene glycol (w/v) for 10 -12 hr. The full-length tr~n~ri~tion products (SEQ ID NO:186) were purified on 8%cl~n~ ring polyacrylamide gels, suspended in TBS buffer [100 mM Tris-HCl, (pH 7.5) 150 mM NaCl) ~binding buffer), heated to 70 ~C, chilled on ice, then incubated with IL-10 at 37 ~C for 10 min. The RNA-protein n~i~l~c was filtered t]hrough a pre-wet nitrocellulose filter then washed with S mL of the binding buiEfer. Bound RNAs were 0 eluted from the filter and lcco~ ed by ethanol pl~ cipil~lion. The RNA was reverse transcribed by avian myeloblastosis virus reverse ll~sc,i~l~se (Life Sciences) at 48 ~C for 45 min with 5'-GCCTGTTGTGAGCCTCCTGTCGAA-3' primer (Table 9, SEQ ID
NO:188). The cDNA w~ amplified by PCR (with 5' and 3' primers (SEQ ID
NOS:187-188)) and the resllltin~ DNA template was tr~n~çri~ed to obtain RNA for the next round of selection. During the course of SELEX, the comt~ntr~tion of IL-10 was decreased r~ lly from S ,uM to 500 nM to pro~lessiv~ly increase selective pl'eS:~Ule.
The selection process was repeated until the affinity of the t-nrichP~I RNA pool for IL- 10 was ~U~ y increased. At that point, cDNA was amplified by PCR with primers that introduced BarnHl and Hind III restriction sites at 5' and 3' ends, respectively. PCR
2 o products were digested with BamHI and Hind III and cloned into pUC 18 that was digested with the same enzymes. Individual clones were screened and sequenced bystandard techniques.

C. Determination of equilibrium ~ soci~tion constants ~d).
Tnt~rn~lly-labeled RNA transcripts were ~.el,~ed by including [a-32P]ATP in T7 RNA polymerase transcription reactions. Full-length LldllS~ were purified on 8%
denaturing polyacrylamide gels to ensure size homogeneity. Gel-purified RNA was diluted to a concentration of~ S nM in TEM buffer, heated to 80 ~C then chilled on ice to facilitate secondary structure formation. RNA concentrations were kept lower than 100 3 o pM in binding reactions. Briefly, equal amounts of RNA were incubated with varying amounts of IL-10 in 50 ~L of TEM buffer for 10 min at 37 ~C. RNA-protein mixtures were passed through pre-wet nitrocellulose filters (0.2 ~L) and the filters were immediately washed with 5 mL of binding buffer. Radioactivity retained on filters was determin~d by liquid sçintill~tion counting. The quantities of RNA bound to filters in the absence of protein was ~lete~ ;..P~l and used for background correction. The percentage of input RNA
retained on each filter was plotted against the corresponding log protein concentration. The nonlinear least square method to obtain the dissociation col~l~ll (K~).

D. Sandwich ELISA
Sandwich ELISA was carried out by using commercially available ELISA kit for 1 0 ~ i ve (1~f ~ I l li l~i ~ ion of hIL-10 (from R&D systems) according to m~nllf~r,turer's instructions. Varying amounts of RNA 43, random pool RNA and anti-hIL-10 monoclonal antibody (from R&D Systems) were inrllh~ted with 125 pg/mL hIL-10 at room te~ )cl~ for 10 min before added to microtiter wells.

EXAl\IPLE 6. 2 -F-MODIFIED RNA LIGANDS TO IL-10 Under nitrocellulose filter binding conditions the random sequence pool that wasused to initiate the SELEX experiment did not show detect~hle binding to IL-10 as high as S ~M conct;llll~lion. However, after twelve rounds of affinity selection the enriched pool exhibited improved affinity, and further selection beyond the 12th round had no effect on increasing the affinity for IL-10. Table 10 (SEQ ID NOS:189-205) shows the sequences identified from the 12th round pool. Sequences are grouped into three classes based on the sequence similarity. The 5' part in the variable 40 nucleotide region of most sequences in class I has sequence complement~rity to the 3' part, suggesting that such sequences can fold into a stemloop structure.
Individual clones were initially screened for their ability to bind IL-10 at 250 nM
concentration. The results show that 20-40% of input individual RNAs was bound to IL-10 at 250 nM. Based on prel i, . ,i"~ y screc..illg, sequence 43 (SEQ ID NO: 189) was chosen as a representative ligand to carry out in section B below.
The Kd of sequence 43 for binding to IL- 10 is 213 nM. The ligand 43, on the 3 o other hand does not bind to other cytokines such as interferon g and IL-4, indicating the specificity of SELEX-derived RNA sequence. Human IL-10 (hIL-10) and mouse IL- 10 W O 96/40717 PCT~US96/09537 (mIL-l0) have high degree of sequence homology at the cDNA and amino acid level (73%
amino acid homology) and hIL-l0 has been shown to active on mouse cells. However, ligand 43 does not bind to mIL-l0 with high affinity.

B. RNA in IL 10 ELISA
An anti-ILl0 monoclonal antibody that neutralizes the receptor binding is commercially available. The R&D systems' Qu~ntikine Tmmuno~s~y kit is based on 96 well microtiter plates coated with the neutralizing antibody to capture hIL-l0. The ELISA
was used to investigate whether RNA binds at or near the neutralizing antibody bindi~ng 1 0 site on IL-l 0. RNA 43, similar to the random pool RNA (used as a control) did not show any inhibition of IL-l 0 binding to anti-IL-l0 antibody on the plate (data not shown).
These data sug~est that the evolved RNA ligand does not bind to the site at or near that recognized by the neutralizing antibody. The soluble anti-ILl0 that was used in the assay as a control behaved as expected, competin~ for binding wit'h the same antibody on the solid phase.

EXAMPLE 7. EXPERIMENTAL PROCEDURES FOR LIGANDS TO hTNF~
This Example provides general procedwres followed and inco,~o~aled in Examples 8- l l for the evolution of nucleic acid ligands to hTNF~.
A. Materials Recombinant hwman TNF~ (hTNF~ ) was pwrchased from Genzyme (C~mhriclge, MA) or R&D Systems (Minneapolis, MN), recombinant mw ine TNF~ (mTNF~), recombinant human TNF~ (hTNF~), and soluble hwman TNF receptor 2 (sTNF-R2) were 2 5 purchased from R&D Systems. Acetylated, and nuclease free bovine serum albwmin (BSA), ligase and restriction enzymes were from new F.ngl~llfl Biolabs (Beverly, MA).
AMV resverse transcriptased were from Life Sciences (St. Petersbwrg, FL). RNasinribonuclease inhibitor, and Taq DNA polymerase was from ]'romega (Madison, WI).
UILI~U1~, nucleotide triphosphates were from Ph~m~ria (Piscataway, NJ). '25-I-TNF~, 3 o ~-32P-ATP, and y-32P-ATP were from DuPont NEN Research Products (Boston, MA).
U937 cells were from ATCC (catalog number CRLl593). Oligonucleotides were obtained W O 96/40717 PCTrUS96/09537 from Operon, Inc. (~l~m~ CA). Nitrocellulose/cellulose acetate mixed matrix (HA), 0.45,um filters were from Millipore (Bedford, MA). Ch~mir~l~ were at least reagent grade and ~ulcllased from commercial sources.

B) SELEX
The SELEX procedure has been described in the SELEX Patent Application (see also Tuerk and Gold, 1990; Gold et al., 1993). The starting RNA contained 30 random nucleotides, flanked by 5' and 3' co~ l regions for primer ~n~ling sites for cDNA
synthesis and PCR amplification (Table 11; SEQ ID NO:206). The single str~n~1ecl DN~A
1 0 molecules were coll~ d to double s~n~1ecl by PCR amplification. PCR conditions were 50 mM KCl, 10 mM Tris-HCl, pH9, 0.1% Triton X-100, 3 mM MgCI2, 0.5 mM of each dATP, dCTP, dGTP, and d ElP, 0.1 units/~l Taq DNA polymerase and 1 nM each of the 5' and 3' primPrs. Transcription reactions were done with about 5~M DNA template, 5units/~l T7 RNA polymerase, 40 mM Tris-HCl (pH8), 12 mM MgCl2, 5 rnM DTT, 1 mM
spermidine, 0.002% Triton X-100, 4% PEG 8000, 2-4 mM each 2'0H NTP, and 0.25 ~M
a-32P-ATP (800 Ci/mmole). For 2'F modified transcripts, 2'F-CTP and 2'F-UTP were used instead of 2'0H-CTP and 2'0H-UTP. Two difr~lenL SELEX t~ c~ ents were done. In the first SELEX ~ nt SELEX-A, the protein was immobilized onto nitrocellulose filters and the RNA ligands were partitioned by capture to the irnmobilized protein.
2 0 Briefly, hTNFa was spotted on a nitrocellulose filter (Millipore, HA 0.45 ~m) and following 5 min air drying over filter paper, the nitrocellulose filter was inc~-b~tecl in a 24-well microtiter plate with 1-2xl0~ M radiolabeled RNA for 30 min at room temperature in 500 ~1 binding buffer (BB=10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 0.02% acetylated BSA, 0.02% ficol, and 0.02% PVP). The filter was then washed 2 5 three times for 10 mimltes each in 1.5 ml BB without BSA. Binding and washing was done under rigorous agitation. The RNA bound to the immobilized protein was recovered by phenol/urea extraction and was then reverse transcribed into cDNA by AMV reverse scliptase at 48~C for 60 min in 50 mM Tris-HCI pH8.3, 60 mM NaCI, 6 mM
Mg(OAc)2, 10 mM DTT, 50 pmol DNA primer-l (Table 11; SEQ ID NOS:206-208), 0.4 3 o mM each of dATP, dCTP, dGTP, and d l~ and 1 unit/~l AMV RT. The cDNA was then PCR amplified and used to initiate the next SELEX cycle as described above. In the W O 96/40717 PCTAU~G~3~7 second SELEX c~ llent, SELEX-B, the binding buffer was Dulbecco's Phosphate-Buffered Saline (DPBS) with calcium and m~gn~sium (Life Technologies, GaiLhc~sburg, MD, Cat. No 21300-025) and the protein-RNA complexes were partitioned by filtçring through nitrocellulose/cellulose ~cet~t~ mixed matrix, 0.45 ,~m pore size filter disks (Millipore, Co., Bedford, MA). Nitrocellulose filter bound RNA w~ recovered by phenoVurea PYt~ctioll- The partitioned RNA was then reverse transcribed and PCR
amplified as above and used to initiate the next SELEX cyc] e.

C. Determinaffon of Equilibrium Dissociaffon Constants 0 To partition the protein-RNA complexes, the binding reactions were filtered through nitroce~ lose/cellulose ~cet~t~l mixed matrix, 0.45 ,um pore size filter disks (Millipore, Co., Bedford, MA). For filtration, the filters were placed onto a V~Cuulll manifold and wetted by as~ilaLillg S ml of DPBS. The binding reactions were as~hdled throught the filters and following a S rnl wash, the filters were co~ulLed in a s~intil~tion counter (Becl;,-,~""). Nitrocellulose partitionsing was used for SELEX and for . ",;..;..~ the eql1ilihrium dissociation constants of RNA ligands to TNFcc. RNAligands to TN~a bind monoph~si~-~lly.
To obtain the equilibrium dissociation col~sk~ '; of RNA ligands to TNFcc the binding reaction:
~3 R:~ = R ?
R=R NA
P--Protein KD=dissociation co~l~l is converted into an equation for the fraction of RNA bound at equilibrium:

q (f/2R T)(PT+RT+KD-((PT+RT+KD) ~PTRT)In) 3 0 q=fraction of RNA bound PT-total protein collce.llldlion W O 96/40717 PCT~US96tO9537 R~total RNA concentrahon f=retention efficiency of RNA-protein complexes The average retention efficiency for RNA-TNFa complexes on nitrocellulose filters is 5 0.1-0.2.
The KDS were rl~t~rrnin~cl by least square fitting of the data points using the software Kaleidagraph (Synergy Software, ~ lin~, PA).

D. Cloning and Sequencing 1 o RT-PCR amplified cDNA from the last round of SELEX was cloned between BamHIand HindIIIrestriction sites of pUC18 plasmid (Vieira et al., 1982, Gene 19:
259-268) in MC1061 E. coli (C~c~ b~n et al., 1980, JMol Biol 138: 179-207).
Seq~enl ing was done using PCR products as templates with a commerically available kit (Promega, Madison WI).
E. Receptor Binding Competition Assay A receptor binding co.l.~lilion assay was used to clet~rrnin~ the bioactivity of the RNA lig~ntlc '25I labelled hTNFa at 0.1 nM was incubated in 50 ~1 of binding medium (PBS with 0.5 mM Mg~, 0.2% BSA, 0.02% sodium azide, 1 U/~l RNasin) for 15 min at20 4~C with serially diluted co---~lilors at 104 to 10-l~ M, and lx104/~l U937 cells.
Duplicate aliquots were subsequently removed, centrifuged through 2: 1 dibutyl-phth~l~te:dinonyl-phthS~l~te lllixlule to separate free and bound l25I labelled hTNFa, and the radioactivity in the pellet was measured on a gamma counter.
Nonspecific binding was ~lett?rminPcl by inclusion of a 200-fold molar excess of unlabeled 2 5 TNF.
The inhibition consklllL~, (Ki) of the RNA ligands were determined by a nonlinear c~ .~.,sion analysis of the data using standard techniques. To obtain Ki values the concentration of TNF receptor was ~Csunlecl to be 3.4x 10-~ ~ M and the Kl, of the I NFR interaction of 0.1 nM.

F. Boundary determination For 3' boundary ~ ion, the 6A RNA ligand was 5' end labeled with y - -32P-ATP using T4 polynucleotide kinase. 5' boundaries were established using 3' end labeled ligand with ~-32P-pCp and T4 RNA ligase. After partial alkaline hydrolysis, the 5 radiolabeled RNA ligand was incllh~te~l with hTNFa at 5, 25, and 125 nM, and the protein bound RNA was isolated by nitrocellulose partitioning. The RNA truncates were analyzed on a high resolution d~n~ rin~ polyacrylarnide gel. An ~lk~lin~ hydrolysis ladder and a ladder of r~rlio~ctively labeled ligands t-qrmin~t~(l with G-residues, generated by partial RNase Tl digestion, were used as EXAMPLE 8. RNA LIGANDS TO hTNF~
A. pr~SELEXchar ~ -Nitrocellulose filter binding could not detect any interaction of hTNFa withrandom RNA even at high protein con~çntr~tions. The binding curves were completely 15 flat even up to lO,L~M hTNFa and RNA up to 1,~4M and the e~;",~lrcl dissociation co~ t (KD) iS greater than lo-3 M. No buffer conllition~ were found that improved the interaction of hTNFoc and r~n-lorn RNA.
To ~l~l~. ,,,i,,~ whether hTNFa was binding any RNA ~lt all we used a more sellsiLive technique similar to no,lhwt;~ probing (Bowen et al., 1980). This technique 2 o was used in various studies of protein nucleic acid interaction and aided in the cloning of various DNA binding proteins (Singh et al., 1988). This experiment showed clearly that some random RNA can bind to hTNFa. RNA binding occurred only when the filter waspreviously spotted with hTNFa and and then dried, but not if the filter was spotted with hTNFa and then placed wet in the incubation chamber. The RNA was binding only on the 2 5 filters carrying hTNFa but not on filters carrying BSA possibly because, either not enough BSA was immobilized on the filter or the BSA present in the iincubation mix was competing for available BSA-specific RNA li~n~l~
-W O 96/40717 PCT~US96/09537 B.SELEX.
Two independent SELEX experimente (A and B) were initi~tPd with pools of randomized RNA co~ .;llg about 10 14 unique molecules. The starting RNA and the PCR primer sequences are shown in Table 11.
In the A-SELEX, the protein was immobilized on a nitrocellulose filter by drying.
The protein C~ P filter was incubated in BB (see Example 7) with labeled RNA, then washed, autoradiographed and the bound RNA was recovered by phenol-urea extraction.
For the first round of A-SELEX about 1,000 pmoles of hTNF~ mo~omer was used and the RNA conc~,lL,~Lion was at 2xl0~M. For the subsequent 14 rounds, two di~lelll filters 0 co"~ il-g about 500 and 100 pmoles of hTNFa monomer were incubated in the same chamber col.l;~ il-g amplified RNA from the previous round at about 2x10~M. Only the RNA from the high protein filter was carried to the next round. A steady increase in the signal to noise ratio was observed and at round 15 the signal retained on the 500- and 100-pmole protein filters was 170- and 35-fold above background respectively. For comparison, in the first round the signal was only about 3-fold above background. RNA
from round 15 had a higher affinity for hTNFa with an estim~t~cl Kd of 5x10-5 M,repres~ntinp~ a possible 100 fold improvement over the random RNA. To increase the stringency of the selection, we carried 8 more rounds using filters with about 10 and 1 pmole of hTNF a . For all these subsequent rounds, except for round 20, the RNA from the 2 o 1 pmole hTNFa filters was carried to the next round. Because of high background, at round 20 we used the RNA from the 10 pmoles hTNFa filter of round l9. The signal to noise ratio for these subsequent rounds became worse at each round but nevertheless the affinity of the evolved RNA continued to improve with estim~t~d final Kd of 7x10-7 M, which ,~,~sel,l~ two additional orders of m~gnitlltle improvement. In the final round, we 2 5 could detect signal with 1 0-fold shorter exposure time was detecte~1 and with 100 - fold less hTNFa on the filter.
In parallel with the stringent phase of A-SELEX, RNA from round l ~ of the A-SELEX was evolved using B-SELEX conditions (see below) for 6 more rounds. We desi~n~ted this as C-SELEX. The affinity of the evolved population at the end of3 o C-SELEX was similar to the round 23 population of A-SELEX with approximate Kd=4xl o-7 M.

CA 02223003 l997-l2-0l The evolved RNA from round 23 had not only improved affinity for hTNFa but it was also specific (Table 13). Binding could be detected only with hTNFa.
In the B- SELEX experiment, binding reactions were set in 25-50,ul and after 10 min inc~lb~tion at 37~C it was filtered through a 0.45 ~m HA nitrocellulose filter. For the 5 first round of the B-SELEX, the RNA and protein were at about 4x10-5 M each. Under these conditions only 0.1% of the input RNA was retained on the filter. This was not surprising since the hTNF~-random-RNA interaction is very weak with a Kd too high to measure and probably in the 1 o-3M range. Subsequent rounds were set similar to the first round. By round 8, the background binding of the RNA to the nitrocellulose filters was 0 very high.

C. RNA sequences and Affinitites RT-PCR amplified cDNA from round 23 of A-SELEX and round 6 of C-SELEX
were cloned and sequenced as described in F.x~nnple 7. 37 clones were sequenced from 15 A-SELEX and 36 cloned from C-SELEX. From the total of 73 sequences, 48 were unique (Table 12; SEQ ID NOS:209-255). A unique sequence is defined as one that differs from all others by three or more nucleotides. Of the 47 unique clones, 18 clones could bind to hTNFa with Kd better than 1 ,uM (Table 12). The best ligand, 25A, (SEQ ID NO:233) binds with affinity dissociation constant at about 40 nM. If it is assumed that the random 2 0 RNA binds with a dissociation constant of greater than 10-3 M, then the affinity of 25A is at least four to five orders of m~gnitu(le better than the starting pool.
Using sequence ~li nment and colls~ ed predicted secondary structure, 17 out of18 clones that bind hTNFa could be ~igned into two classes.
The members of the class II can be folded in stem-loop structures with internal 2 5 bulges and asymmetric loops. Linear sequence ~ nment did not reveal any significant conserved sequences.

D. Speeifi~ity of RNA l i~ to TNF
We tested the specificity of the evolved pool of round 23 of A-SELEX against 3 o human TNFa, human TNFB and murine TNF~. The evolved pool is highly specific for human TNFa and specificity ratios are shown in Table 13.

W O 96/40717 52 PCT~US96/09537 EXAMPLE 9. Inhibition of hTNFa Binding to Cell Surface rcce~lors To test the ability of the TNFa ligands to competitively inhibit the binding of hTNFa to its cell surface receptor, the U937 cells were used to sçreen several hTNFa lig~n-1c The observed Kis are listed in Table 14. The data show that several ligands can competitively inhibit binding of hTNFa to its cell surface receptors while random RNA
cannot. Ligand 25A has the highest potency with a Ki of 21 nM. This Ki value is only 6 fold worse than the Ki observed with the sTNF-R2 under the same ~ llental conditions.

EXAlViPLE 10. ~ffect of 2'F Pyrimidine ModificaHon on the Binflin~ and Inhibitory Activities of the ~TrnFa T.i~an~c Tl~lscli~l~ co~ g 2'F modified pyrimitlines are resistant to RNase degradation. To obtain ligands with improved stability we tested the effect of 2'F
pyrimidine modification on the binding and inhibitory activity of several hTNFa lig~ntlc The results sllmmz~ri7~1 in Table 15 show that some of the ligands retained binding activity when are modified with 2'F pyrimidines but in general the modified ligands bind worse than the unmodified c~ ~L~. Class II ligands are in general more tolerant of the 2'F pyrimidine modification. Most of the ligands that retained binding after the 2'F
2 o pyrimidine modification lose their inhibitory activity. Only the 2'F pyrimidine modification of the most abundant ligand, 6A, did not affect its binding and inhibitory activities.

EXAlVIPLE 11~ tal Proc~lur~,s for DNA T.~-~lc to R.ANTF~
2 5 This example provides general procedures followed and incorporated in Example 12 for the evolution of nucleic acid ligands to RANTES.

A. Materials Recombin~nt human RANTES was purchased from Genzyme (Cambridge, MA).
3 o Taq DNA polymerase was Perkin Elmer (Norwalk, CT). T4 polynucleotide kinase was purchased from New Fn~l~n-l Biolabs (Beverly, MA). Ultrapure nucleotide triphosphates were purchased form Pharmacia (Piscataway, NJ). Affinity purified streptavidin (Cat. No 21122) was from Pierce (Rockford, IL). Oligonucleotides were obtained from Operon, Inc. (Alameda, CA). Nitrocellulose/cellulose acetate mixed rnatrix (HA), 0.45 ~m filters were purchased form Millipore (B,edford, MA). Chemicals were at least reagent grade and 5 purchased from commercial sources.

B. SELEX
The SELEX procedure has been described in detail in the SELEX Patent Applications. The DNA template cont~ned 40 random nucleotides, flan~ed by 5' and 3' 0 constant regions for primer ~nt-~lin~ sites for PCR (Table 16; SEQ ID NOS:256-258).
Primer 3G7 (SEQ ID NO:258) has 4 biotin residues in its 5' end to aid in the purification of single str~nclçd DNA (ssDNA). For the first round, 105 prnoles of synthetic 40N7 ssDNA were 5' end labelled using T4 polynucleotide kinase in a 25,ul reaction CO..~ )g 70 mM Tris-HCl pH 7.6; 10 mM MgCl2, 5 mM DTT, 39.5 pmoles of g -32P-ATP (3000 5 Ci/mmol), and 16 units kinase, for 1 h at 37~C. The kinslee~l DNA was then purified on an 8% polyacrilamide, 7M urea, clt?~ gel and then mixed with gel purified unlabeled 40N7 to achieve about 5,000 cpm/pmol specific activity. To prepare binding reactions, the DNA molecules were incubated with recombinant RANTES in HB' B~l~n~e~l Salt Solution OEIB,SS) without calcium and m~ e;ul,l (Life Technologies, Gaithc.~b~u~,, MD, 20 Cat. No 14175) co..l~t;..i..~ 0.01% hurnan serum albumin. Two SELEX experim~nte were performed, one with normal salt concentration and the other with 300 mM NaCl. The high salt concentration was achieved by adding additional NaCl to the HBSS. Followingincubation at room temperature for 30 ...;....lee the protein-DNA complexes werepartitioned from unbound DNA by filtering through HA nitrocellulose 0.45 ~m.
2 5 Nitrocellulose filter bound DNA was recovered by phenol/urea extraction. The partitioned DNA was PCR amplified in 50 mM KCl, 10mM Tris-HCl, pH9, 0.1% Triton X-100, 3mM
MgCl2, 1 mM of each dATP, dCTP, dGTP, and d l TP, with 0.1 units/,~l Taq DNA
polymerase. The 3G7 and SG7 primers were present at 2 ~M. The 5G7 primer was 5'-end labeled before use described above. To purify ssDNA, the PCR product was ethanol3 o precipitated and then reacted with affinity purified streptavid;n at a molar ratio 1: 10 DNA
to streptavidin in 10 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% sodium azide. Following 30 inc~lh~ti- n at room te~ c;ldLule, equal volume of 100% follll~LIllide tr~ ing dye was added and the strands were denatured by incubating at 85~C for 1.5 min.
The denatured strands were then electophoresed in an 8% polyacrylamide, 7M urea gel and the non~hi~ed band was excised and purified from the crushed gel. The purified 5 ssDNA was then used for the next SELEX cycle.

EXAMPLE 12: DNA LIGANDS TO R~NTES

A. SELEX
To generate DNA ligands for RANTES, two SELEX ~x~clhllents were performed, one with 150 mM and the other with 300 mM NaCl. The high salt was used in order to avoid ~lccipiLdlion of t_e RANTES-DNA complexes that occurs at the lower salt concentration. The SELEX at 300 mM salt was prematurely termin~ted because of high background. The SELEX conditions and results for each round of the 150 mM salt 5 SELEX are sllmm~ri7P~l in Table 17. The starting pool colllailled 1.8x10~5 (2,940 pmoles) of DNA for the 150 mM salt SELEX. The starting KD values of the random DNA were 3xlO~M. After 19 rounds of SELEX the evolved pools bound with a KD of 20 nM. This represents about 150 fold improvement.

W O 96/40717 PCTrUS96/09537 Table 1 40N7 TEMPLATES AND PRIM[ERS
SEQ ID
NO.
40N7 ssDNA Template:
S' GGGAGGACGAUGGGG[-40N-]CAGACGACUCGCCCGA 3' SELEX PCR Primers:
5P7:
5' TAATACGACTCACTATAGGGAGGACGATGCGG 3' 2 3P7:
5' TCGGGCGAGTCGTCTG 3' 3 Cloning PCR Primers:
5P7H:
Hind III
S' CCGA~GCTTAATACGACTCACTATAGGGAGGA~"GATGCGG 3' 4 3P7B:
Bam HI
5' GCCGGATCCTCGGGCGAGTCGTCTG 3' S
PCR Sequencing Primer:
pUC19F3 0:
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3 ~ 5 ~ _ p ;~ J ~ J C' ~.' m ~ ~ ~ _ 3 0 ~ ~ r-0 J J ~ a v J C ~ V _ _ ~-a ~ ~ ~ _ o v J C, ~r V ~ 0 ,5 o rl ~ J ~ _I -- ~ ~ ~ a _ u ~ C ~ C, O J ~q J ~ C O _~ ~ O
V r C ~1 ~7 V 3 ~
J P
~ 0'1 ~I J ~-C 3 N ~ ;~
~ J 1 C N ~ V
O a~ O~D 0 ~
O ~cn _I _I N ~ ~r ~1 _I N N ~ I H
~ re ~ ~ # ~ ~ e o ~ Z

H
o t~ ao a~ o ~1 ~ ~ d' L.
~n ~0 w P~ .
~ .
r~ r~ ~ r~ r~ ~ r~ J
r r ~ rr r r i~ c r~ ci ~ c r_ r~ J J _ ~ J r' J ~ ~ ~ ~ ~
~ r; ~ ~
~ r .-- 3 ~ r ~r ~ ~C r_ _ ~

J ~ r r~ rc ~ ~ 3 r r~
r- C r V r; ~ r ~ C ~ ~
v ~ ~ r ~ ~ ~ c r_ i~ - r~ r r ~ r_ r~ ~ C r~ C
i~ r; ~ ~ ~ r r ~ c ~ r_ r_ c ~ r_ r ~ c - ~ r r ~ ~ J C r~ c ~ ~ r r r; ~ r~
J ~ i J ~ r~
r - ~ r~ ~ c ~ J r_ ~: r ~ ~ r_ r~
~r ~ ~ r ~ r v r ~ ~ r_ r~ ~ ~ r_ ~ r~ ~ ~ r~ ~
r C ~ r_ r ~, J _ C P
~5 ~ ~-3 ~ 5 r5 r, ~ ~1 ~ H N d~ Ll') ~
###

CA 02223003 l997-l2-Ol W O 96/40717 PCT~US96/09537 H
O ~ ~~ O .
~n _ g r~ v ~ 1 v v v ~ ~ v US
- ~ 8 ~ r~ ~ ~ ~v _I ~ _ _ D _ ~ ~-1 D ,,~ ~ r~
~1~ ~ g ~ ~ . f ~ r~ ~ 1 ~ or~ ~
r~
r~ ) o . ~c a ~ r r u 1I r~
c ~ tn ~ ~ ~ u ~ ~:n ~o ~ ~ V
-- ~ Z
C~ 1 -- . I -- N ---- N -- -- O' - r l _ N _ E # It7 x In # IJl # 1~'1 ~ # u) :te 11) X u~ # 11'1 W O 96/40717 PCT/U~,G~5537 Table 9 SEQ ID
NO.
Initial r~n~ ~eguence RNA pool:
5'-GGGA~ U~UUU~ACGCUCAA-(N)~o- W CGACAGGAGGCUCACAACAGGC-3' 186 5'-Primer:

5'-TAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3' 187 3'-Primer:
s~-Gc~l~Ll~l~AGc~lc~l~l~AA-3~ 188 W O 96/40717 PCTrUS96/09537 Table lO
SEQ ID
NO.
43(10): ACAUCGUAUAA---CUCUA-AGGGCCUGGAUAUACGAUGAA 189 64 : ACAUCGUAUAA---CUCUA-AGCGCCUGGAUAUACGAUGAA 190 64a : ACAUCGUAUAA---CUCUA-AGAGCCUGGAUAUACGAUGAA 191 64b : ACAUCGUAUAA---CUCUA-AGUGCCUGGAUAUACGAUGAA 192 55 5 : ACAUCGUAUAAU--CUCUA-AGAGCCUGGAUAUACGAUGAA 193 68 : AcAucGuAuAAu~:u~:u~:uA-AGAGccuGGA-AuAcGAuGAA 194 6 : AUCCCA-AU---CUCUA-AGAGCCUGGA-U---AAGAAUGCGCAWGGGC 195 54 : AUCCCA-AU---CUCUA-AGAGCCUGGA-U---GACAAU-CGCAWGGGC 196 57 : AUCCCA-AU---CUCUA-AGAGCCUGGA-U---GAGAAUGCGCAWGGGC 197 : CUGAGAU---CUCUA-AGAGCCUGGACU-CAG-CUCCGACUGACC 198 34 : CUGAGAU---CUCUA-AGAGCCUGGACU-CAG-CUCCGAWGAUCC 199 41 : CUGAGAU---CUCUA-AGAGC~u~ACU-CAG-CUCCGAWGAACC 200 15 (2) : UCUCUA-UGAGCCUGGA-U-CGACGAA~:u~:u~uACGGGCUGUG 201 56 (2) : UCUCUA-AGAGCCUGGA-U-GUCGAGGGGCCAWWCGCACGC 202 2 : AUCUCUACUGAGCCUGGA-U-UCGCCAGAAGWWAUCACAGU 203 59 : CGUA~AAGWAUCGAAU--CUCUG-UGAGCCUGGA-U-CGAWAC 204 3 :CUGAGAU---CUCUA-AGAGCCUGGACUCAGCUACGAWGAGC~uuuAWCWG 205 W O 96/40717 PCT~US96~'093~7 Table 11 SEQ ID
" NO.
STAK~ N~ RNA:
5'-GGGAGCUCAGAAUA~ACGCUCAA (N30) W CGACAUGAGGCCCGGAUCCGGC 206 PCR PRIMER 1:
Bam~ I ~
5~-GCCGGAlCC~GGC~L~ATGTCGAA 207 PCR PRIMBR 2:
T~ n~l III
5~-CCGAAGCTTA~T~C~.~CT~CTAT~GGGAGCTCAGAATA~ACGCTCAA 208 T7 promoter o ~1 ~ r'~ ~ 0 o~ o rt t'~ ~ ~ 1~ ~ r- ~ a~ O r-l t~
H o ~I r-l ~I r-l _I ~I r-l r-l ~1 ~1 ~ ~ t~ ~ ~ t~ ~ r.
O~ O ~ ~ ~ r~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N ~ ~ ~ ~ ~ ~ r.~ ~ ~ ~ ~ N
~i3 Z
cq H H H H H H
U~ O O U~
r.~ rD N O
r ,1 ~ ,1 , , , , ~ , , ,., , , , , . - - ~ ~ ' ? ' ' ' ' ' ' ' ' . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . - .
. . . . . . , , , , , , , , , , , , , , , , , , , , , , ~ - - - - - - - - - - - - - - - - - . . . . . . . . . .
~ . . . . . . . . . . . . . . . . . . . . . . . . . . . .
~ .
u V ~C E- U, V J ~ v U r~ U - ~ rJ ~ r~
- - ~ U U ~ U E~ U ' U - ~ U E~ - U - ~ U
~ r ~ r~ g _ ~ ~ r r ~U
~I ~ r - ~ - ~ - ~ ,, rJ _, ~
) r ~ _ ~ ~ r~ , r ~ V ~~ r.~ r r a r_~ J ~ J j~ r U - r t ~ 5 J ~ g ~ r r~
E- ~C~ r' ~ _ c I ~¢ t - ~, _ - 5 r ~ I c ~ ' ~ ~ ~ J r J r r~ ¢ - ~ C~ C r _ _ r ~ r ~ r r~ ' r.
~i! 5 -5~ ~UC r C~ ~ - U~ r~
C~_ U ~ C~ _ ~ r r El r~ ? r~ ~ r~ -t ~ 5 l ' 5 ~ 5 r~ 5 - t ~) I t . 5 ~;
r~ rr~ t' _ _ ~ l: _ . J _ ~1 V ~ J l _ r~ J ~ ~ _ _~ J
~ ~ r ~ J
-2~
. . . . . . . . . . . . . . . . . . ~ . . . . . . . . .
I~

m u o~ t' ~ ~ ~~ -- -- --~U r~ ~S ~¢ ~S ~ 4 ~¢ O ~ r 1~ 0 1' CD ~ O r-~ N r~ rD
rn r~ ,~ r-~ _~ r-~ r-~ _~ r-~ ~ r-l ~ ~ ~ r. ~ ~ ~ ~ ~ ~

W O 96/40717 PCT~US96/09537 t-- 0 a~ o ,-1 ~ t' ) ~ In LD 1-- L~ a~ o r1 t~l ~ ~ u~ S-l O ~ ~1 ~ (~ L~ N L 'l ~ ~ ~ t~ N ~'1 t~ ~ ~ _ 01 Z ' 5 ~ a~

~ cn ~ ~ O ~ 0~. 0 ~r o o --I ~ N ~ r~l In _I ID IS) r~
;
U I I ~ ~ O
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ~ ~ V
O
. ~ r~
~ ~ ~ ~ ~ ~~ LU ~ O
a p, ,_ V
r t I V ~
'r . . . . . . . . . . . . . . . . . . ~
. . C O
O ~ n ~ _ ~ ~ . ~ . . ~ V O V
V ~ ~ rJ ~ rJ ~ rJ r- ~ 1 :J r~ n) O
r~ _ V v _ ~: v, ~ - ~ r~
_ v ~~ rJ ~ v r r V rJ ~ ; ,r ) ,~ , C ~, s ~
m ~t ~ c 6 ' Et V ,~ t ~ ~ O
V ~ V V ' J ~ ~J ~ ~ - _ V.0 ~ U V V ~ V :~ ~ V ,a ~ ~ ~o c. 1'1: _ V ~ _ ' V '5 ~ ~~ ~ Et _ ~r ~ ~ 1 r . ' ~ ,D~ ErJt ', ~ - ' ED ~ . ~ '' e,~ a~
V - ',: J ~ ~ - - ~ ~' v n~
E~ - - ' ~
! ' a~ ~ a v~
3 ~, v .~ ~ - 3 ~ ~ ~ ~ -Z v 0 _ ~è ~~
Vl ~I . O
a) ml -- _ . . . . . . . . . . _ . .
S r3 -~
~) U n~ ~ , tr~ e m O u~- - - - - - tr U _ ~ a ~, ~ ra) ~'~ ~ ~~ V V V V V C~ V V V ~ c s~ ~~ S O
a)_~ v v v v v v v v v O ~ r Ln ~ ~ O Q V ~t Et Z
rn v ,~ D r~ E ~:1 .a t ~ct W O 96/40717 PCT~US96/09537 B;n~;ng specificity of the evolved pool of ligands from round 23A

Ratio:
T~rget ~n, ~ ~Target /~hT~
hTNF~ 700 hTNF~ >1,000,000 ~1,400 mTNF~ ~1,000,000 ~1,400 CA 02223003 l997-l2-Ol W O 96/40717 PCT~US96/09537 T~iBLE 14 Ki values of hTNF~ competitor~ on the U937 cell competition a~ay Com~et;t;or K;. ~ R~
~TNF-R2 3.3 0.99323 random RNA ~1,000,000 6A 9,100 0.93776 25A 21 0.98105 4C 1,200 0.93496 14C 930 0.88453 18C 2,500 0.97483 ~Fit correlation coefficient W 096/40717 PCT~US96/09537 Effect of 2'F-pyrimidine modification in the affinities Y
and inhibitory activities of the hTNF~ ligands 2'OH 2'F 2'0Ha 2'F
Clo~e K~. n~ K~ K~ Ki,n~ Cl~ss 3A (2) 135 623 4C 92 NBb 1, 200 7C (2) 297 442 8A 785 ~ c llC 140 ~

16C 200 123 -313 9,191 l9C 83 NB
23A 52 400 -241 14,671 25A (2) 40 445 21 6A (7) 120 74 9,100 8,156 II
8C (2) 430 503 II
14C 60 133 930 11,540 II
18C 460 NB 2,500 O
aKi values were obtained based on 5-8 point curves except for 16C and 23A 2'OH ligands where only 3 points were used.
bNo binding.
~Not determined.

Table 16 SEQ ID
NO.
Starting DNA:
4ON7:
5'GGGAGGACGATGCGG[-4ON-]CAGACGACTCGCCCGA 3' 256 SELEX PCR Primers:
5G7:
5'GGGAGGACGATGCGG 3' 257 3G7:
5'XXXXTCGGGCGAGTCGTCTG 3' 2 58 X~biotin o o a~
o a~
r7 ~ rl o o o ~r ~ o o D O O O O O O O O O O
o ~ Lrl o ~ o ~
O ~D ~ O CO a~ r U

m Ir) o o o o o o o o o p ~ ~ ~ ~ u~ ," ~

,., :
~ 3 E ~ ~ ~ ~ ~ o o o o o o o o o ~ a ~ ~ N ~ ~ ~ U~ o ~ m ~ ~ h ,~ tn ~ + + + + + + + + + + + + + + + + + + + a ; L~
U tl5 ~4 3 ~
~

" ~ LOl ' ;
g ~ ~ N ~r r~ U') ~) ~ .~ ~ ~ ~' U~ t~ N ~ _ ,~ L) W ~ ,~ o o o o o o o o o o ~i o o o o ~ ~ ~ ~ '' h L) o a~ h -~1 ~ O O O O O O O O O O O O O O O ~ ~ ~ ~ ', Z L) ~ L) e o ~ o o o o o o o o o o O o o ~ ~ ~ ~ a) ~) 3 'O ~

~ 40 j, o o o o o o o o o O O O O O O O ~0 a r ~ u-~ ~ 'v o o o o o o o o o o o o o o o o o o o ~ a - ~ ~
~ ~o ~ o o o o o o ~ o r N O ~ ~ ~ O O 0. a o o L ~

V ~t 1~ ~ ') N ~t ~ N N N 0~ W C~ _ . _ ~
O ~~ O .t N ~ ~ ~ ~ r ~ ~ ~ ~ ~ ~ ~ ~
t ~t N Iq ~ Ul W 1' ~ 0~ ~t ~1 ~t ~t ~t ~1 ~t ~t ~t ~t ~, ~ C~

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TCGGGCGAGT CGTCTG l6 (2) INFORMATION FOR SEQ ID NO: 4:
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(i) SEQUENCE CHARACTERIZATION:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CA 02223003 l997-l2-0l (2) INFORMATION FOR SEQ ID NO: 6: o (i) SEQUENCE CHARACTERIZATION:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

(2) INFORMATION FOR SEQ ID NO: 7:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

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(i) SEQu~N~ CHARACTERIZATION:
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WO96/40717 PCT~S96/09537 (ix) FEATURE:
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- modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQu~N~ CHARACTERIZATION:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQu~ CHARACTERIZATION:
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modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 11:
GGGAGGACGA UGCGGACACC G W AAUCUGA GGCCCUGUCC U~W CCUCCA 50 (2) INFORMATION FOR SEQ ID NO: 12:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQu~N~ CHARACTERIZATION:
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(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 71 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GGGAGGACGA UGCGGACACC G W A~ACUGA GGCCCUGUCC UA W CCW CA 50 (2) INFORMATION FOR SEQ ID NO: 15:
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(ix) FEATURE:
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modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GGGAGGACGA UGCGGAACAC CCCCG~u~u~ ACG~uu~uuC CGAA W CCUC 50 CAC'C~uCAGA CGACUCGCCC GA 72 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERIZATION:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (A) LENGTH: 73 base pairs (B) TYPE: nucleic acid - (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
Y (ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
GGGAGGACGA UGCGGGAACA CCCCCGGUCU GACG~uu~uu CCGAA W CCU 50 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQ~N~ CHARACTERIZATION:
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(ix) FEATURE:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GGGAGGACGA UGCGGAACAC CCCGGUCUGA CG~uu~uuCC GAA W CCUCC 50 (2) INFORMATION FOR SEQ ID NO: 18:
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modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
GGGAGGACGA UGCGGAACAC CCCCGGUUUG ACG~uu~uuC CGA~AUCCUC 50 (2) INFORMATION FOR SEQ ID NO: 19:
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(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l W096/40717 PCT/U',G/09~37 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEgu~ DESCRIPTION: SEQ ID NO: 19:
GGGAGGACGA UGCGGAACAC CCCCGGUCUG ACG~uu~uuC CGAAUCCUCC 50 (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
GGGAGGACGA UGCGGGG W C ~U~UU~UACU W CUAA WAU CCGCACCUCC 50 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERIZATION:
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(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GGGAGGACGA UGCGGGA W C A W W GAUCU uu~uuuCUCU ~AUCCCGCUG 50 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQu~N~ CHARACTERIZATION:
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(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 23:
GGGAGGACGA UGCGGA W CC uuuuuCCU W ~u~uuUu~uG ACCGACUGAU 50 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GGGAGGACGA UGCGGUAAUC UACAC W AUA uuuuuuuu~u u uuu~uu uC'C 50 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQu~N~ CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
GGGAGGACGA UGCGGAGGGU UGGGAGGGGU CCUU~uuuuC GUCUGCGUGG 50 (2) INFORMATION FOR SEQ ID NO: 26:

(i) SE~N~ CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear W O 96/40717 PCT~US96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
- (D) OTHER INFORMATION: All pyrimidines are 2'-NHz modi~ied - (xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 29:

(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 7Q base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 31:
GGGAGGACGA UGCG~Uu~uA GCGCGAUAUA GCGCUGGUAG GG W GCCGGU 50 (2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied WO96/40717 PCT~S96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(iX) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 34:
GGGAGGACGA UGCG~uu~uA GCGCGAUAUA GCGCUGGUAG GG W GCCGGU 50 (2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) S~u~N-CE DESCRIPTION: SEQ ID NO: 35:
GGGAGGACGA UGC~u~uA GCGCGAUAUA GCGCUGGUAG GG W GCCGGU 50 (2) INFORMATION FOR SEQ ID NO: 36:

WO96/40717 PCT~S96/09537 (i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GGGAGGACGA UGCG~uu~uA GCGCGAUAUA GCGCUGGCAG GG W GCCGGU 50 (2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 69 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 5 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

CGGUGCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 40:
GGGAGGACGA UGCG~u~u~u GGGGUGCCAU AUAACCCCGG W GGG W GAC 50 G~u~U~AGAG CGACUCGCCC GA 72 (2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

(2) INFORMATION FOR SEQ ID NO: 42:
(i) S~u~NCE CHARACTERIZATION:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRAND~nN~S: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

(2) INFORMATION FOR SEQ ID NO:43: .
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l;ne~r (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GGGAGGACGA UGCGGAGGCU CA~AAGGCCG G W GGG W AG GUAA~u~u~u 50 (2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 67 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 71 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

(2) INFORMATION FOR SEQ ID NO: 46:

WO96/40717 PCT~S96/09537 (i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NHz modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlU~E:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQU~N~ DESCRIPTION: SEQ ID NO: 47:

(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NHz modi~ied (xi) SEQU~ DESCRIPTION: SEQ ID NO: 48:

AGGGUCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO: 49:
(i) ~Qu~ CHARACTERIZATION:
(A) LENGTH: 71 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GGGAGGACGA UGCGGUAUAG GUAACUAUCA GGUGGGUAGU CGGUGGA~AC 50 GGG~u~uu~G UCAGACGACU CGCCCGA 77 (2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERIZATION:
-(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 pyrimidines are 2'-NH2 modified (xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 51:
GGGAGGACGA UGCGGUACAG GUGG~u~uG GAUAA W GGG CACGCUCUAU 50 (2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified WO96/40717 PCT/U~3G~9537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
GGGAGGACGA UGCGGCACUA GGUGGGUCGU G~uu~uu~GG CACGUAAC W 50 CGCGUCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
GGGAGGACGA UGCGGUACUA GGUGGGUCGU G~uu~uuGGG CACGUAAC W 50 CGCGUCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQu~ CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

UCAGUCAGAC GACUCGCCCG A 7l (2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-NH2 modi~ied (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 57:

(2) INFORMATION FOR SEQ ID NO: 58:
(i) SE~u~N~ CHARACTERIZATION:
(A) LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEAlu~E:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERIZATION:
(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 U' 6 are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 71 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQu~ DESCRIPTION: SEQ ID NO: 60:

(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERIZATION:
(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 U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA

W O 96/40717 PCT~US96/09537 (ix) FEATURE:
(D) OTHBR INFORMATION: All U' 8 are 2'-MH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQ~N~ CHARACTERIZATION:
(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 U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQ~hN~ DESCRIPTION: SEQ ID NO: 63:
GGGAGGACGA UGCGGCCAUA GUGGGUGGGU W GGAGUGGA A.UAGUGCCGA 50 (2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERIZATION:
(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 U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERIZATION:
Q (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 U's are 2'-NH2 modified CA 02223003 l997-l2-0l (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
GGGAGGACGA UGCGG W CCG UCCGUGGGAU AG~uuu~uGG GAUGUACCGG 50 (2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQD~N~ CHARACTERIZATION:
(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 66:

(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 46 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE: L
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

CA 02223003 l997-l2-0l WO96/40717 PCT/U~,G/0~S37 (2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
GGGAGGACGA UGCGGAACAC CCCCGGUCUG ACG~uu~uuC CGAA W CCUC 50 (2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECUhAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
GGGAGGACGA UGCGGAGGGU UGGGAGGGGU C~UU~UU 37 (2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 51 ba8e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GGGAGGACGA UGCGGUGGUA GCGCGAUAUA GCGCUGGUAG (,G W GCCGGU 50 (2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear CA 02223003 l997-l2-0l (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:

(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
GGGAGACAAG AATA~ACGCT CA~NNNNNNN NNNNNNNNNN ~NNNNN~NN~ 50 (2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:

(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid -WO96/40717 PCT~S96/09537 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOhECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 5 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 77:

(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRAN~h~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
GCCGGATCCG C~l~ll~l~A GCCTCCTGTC GAA 33 (2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRAN~:~N~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
GGGAGACAAG AAUA~ACGCU CAACUAUGGG GAGCCACA W A~CGGCAAUA 50 (2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 89 base pairs (B) TYPE: nucleic acid (C) STRANDEDNBSS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 80:

(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 ba6e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:

AUAUAA WW A ~uuuC~ACAG GAGGCUCACA ACAGGC 86 (2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:

(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQ~ CHARACTERIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified WO96/40717 PCT~US96/09537 (xi) SE(,~J~N~; DESCRIPTION: SEQ ID NO: 83:

(2 ) INFORMATION FOR SEQ ID NO: 84:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 88 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULAR TYPE: RNA
( ix ) FEATURE:
(D) OTHER INFORMATION: All pyrimidines arè 2 ' -F
modified (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
GGGAGACAAG AAUA~ACGCU CAAUCCCACC GGGGUGCCAC GGWWA~ACG 50 (2) INFORMATION FOR SEQ ID NO: 85:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( i i ) MOLBCULAR TYPE: RNA
( ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85::

( 2 ) INFORMATION FOR SEQ ID NO: 8 6:
( i ) SEQUENCE CH~RACTERIZATION:
(A) LENGTH: 8 7 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULAR TYPE: RNA
( ix) FEATU~E:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -F
modified (xi) SEQu~;N~ ~; DESCRIPTION: SEQ ID NO: 86 ::

AUAUAAWW A~ :uuuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:

(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQU~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATU~E:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:

(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQU~N~ CHARACTERIZATION:
(A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) S~Q~NCE DESCRIPTION: SEQ ID NO: 89:

(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified ~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
GGGAGACAAG AAUAAACGCU CAACUAUGGG GAGCCACA W ~AACGGCUAU 50 (2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
GGGAGACA~.G AAUAAACGCU CAAACUGGGG AGCCA QGAU UUAACGGCGC 50 (2) INFQRMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 92:
GGGAGACA~G AAUAAACGCU CAACUCUCAC UGGGGAGCCA CA~uuuuAAA 50 (2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:

(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 94:

(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 89 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi f ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:

(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modi f ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:

(2) INFORMATION FOR SEQ ID NO: 97:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:

(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEA'lUKE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:

(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQU~N~h CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:

GAUGGGAUGC CC~uu~GACA GGAGGCU QC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear WO96/40717 PCT~US96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 100:

(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:

CGAUCUGGAG u~uuC~ACAG GAGGCUCACA ACAGGC 86 (2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:

(2) INFORMATION FOR SEQ ID NO: 103:
(i) ~u~ CHARACTERIZATION:
(A) LENGTH: 80 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 6 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SE5,2u~;N~:~; DESCRIPTION: SEQ ID NO: 103:

(2 ) INFORMATION FOR SEQ ID NO: 104:
., ( i ) SEQUENCE CHARACTBRIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( ii ) MOLECULAR TYPE: RNA
( ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -F
modi~ied (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 104:

(2) INFORMATION FOR SEQ ID NO: 105:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 8 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( ii ) MOLECULAR TYPE: RNA
( ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:

AAUAGGGAUG CC:(~;uuCGACA GGAGGCUCAC AACAGGC 87 (2 ) INFORMATION FOR SEQ ID NO: 106:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( ii ) MOLECULAR TYPE: RNA
( ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -F
modified (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 106:
GGGAGACAAG AAUAAACGCU CAAGCUAACC CGUACAAAW UU~ uuuuuCA 50 (2) INFORMATION FOR SEQ ID NO: 107:

CA 02223003 l997-l2-0l (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) ~QD~NCE DESCRIPTION: SEQ ID NO: 107:

(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNE$S: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:

(2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:

(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear WO96140717 PCT~US96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 110:

AGGGGC W GG GC~uuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
GGGAGACAAG AAUA~ACGCU CAAC~uu~uG GGGAGCCACG ~AUACGGCCA 50 (2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRAN~N~ss: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
GGGAGACAAG AAUAAACGCU CAAGAGCUGG UGAGCCACGU A.UACGGCC W 50 AGGGGC W GG GC~UUCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied CA 02223003 l997-l2-0l WO96/40717 PCT/U~Gl0~537 (xi) SEQUENCE DESCRIPTION: SEQ ID-NO: 113:

AGGGGC W GG GC~uuC~ACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOhECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:

(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:

(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPO~OGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 116:

(2) INFORMATION FOR SEQ ID NO: 117:

( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECUL,AR TYPE: RNA
( ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -NH2 modified (xi ) SEQU~;N~:~; DESCRIPTION: SEQ ID NO: 117:
GGGAGACAAG AAUA~ACGCU CAAUCACAAG CACCCWGGG GAGCCACAW 5 0 (2 ) INFORMATION FOR SEQ ID NO: 118:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( ii ) MOLECUhAR TYPE: RNA
( ix ) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -NH2 modified (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 11~3:
GGGAGACAAG AAUA~ACGCU CAAAUGGAGA GCCACAWAA CGGCAGCAUA 5 0 UCACAGUAGG A~WCGACAG GAGGCUCACA ACAGGC 8 6 (2 ) INFORMATION FOR SEQ ID NO: 119:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNES S: single ( D ) TOPOLOGY: l inear ( i i ) MOLECUIIAR TYPE: RNA
( ix ) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2 ' -NH2 modi~ied (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
GGGAGACAAG AAUA~ACGCU CAAUGUGGGG AGCCACAGW AACGGCWCA 5 0 ( 2 ) INFORMATION FOR SEQ ID NO: 12 0:
( i ) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 t modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:

CAACGGCA W ~u~uuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 121:

(2) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQu~ DESCRIPTION: SEQ ID NO: 122:

CA~CGGCGCA GA W CGACAG GAGGCUCACA ACAGGC 86 (2) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (xi) SEQU~ DESCRIPTION: SEQ ID NO: 123:

CCAC~u~uAC GGC W CGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 124:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:

(2) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 8 6 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECUL~R TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
GGGAGACAAG AAUA~ACGCU CAAGCGGUCU GA W GAGCCA CCGUGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:
GGGAGACAAG AAUAAACGCU CA~ACAA WW CACACAGA~A CAGCUAUGAC 50 (2) INFORMATION FOR SEQ ID NO: 127:

CA 02223003 l997-l2-0l WO96/40717 PCT~US96/09537 (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:

(2) INFORMATION FOR SEQ ID NO: 128:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQU~N~ DESCRIPTION: SEQ ID NO: 128:
GGGAGA Q AG A~UAAACGCU Q AAUA QAU GUGG W GAAG CUACCUCC Q 50 AGuGG GCC W CGA Q GGAGGCU QC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 129:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 129:

(2) INFORMATION FOR SEQ ID NO: 130:
(i) S~Q~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:

(2) INFORMATION FOR SEQ ID NO: 131:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:

(2) INFORMATION FOR SEQ ID NO: 132:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132::
GGGAGACAAG AAUA~ACGCU CAAUCGAUAC UACUCCUGGA GA~AAGGGAG 50 (2) INFORMATION FOR SEQ ID NO: 133:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified CA 02223003 l997-l2-0l WO96/40717 PCT~US96/09537 (xi) SE~ ~ DESCRIPTION: SEQ ID NO: 133:

(2) INFORMATION FOR SEQ ID NO: 134:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATuKE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:

(2) INFORMATION FOR SEQ ID NO: 135:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:

(2) INFORMATION FOR SEQ ID NO: 136:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) S~Q~NCE DESCRIPTION: SEQ ID NO: 136:
GGGAGACAAG AAUAAACGCU CAAUCGAUAC UA~u~u~GA GAAAAGGGAG 50 (2) INFORMATION FOR SEQ ID NO: 137:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NHz modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:
GGGAGACAAG AAUA~ACGCU CAACCGAUAC UACUCCUGGA GA~AAGGGAG 50 (2) INFORMATION FOR SEQ ID NO: 138:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:

(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlu~E:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
GGGAGACAAG AAUAAACGCU CAAUCGUAGC CUCCAGCGGA ~UGCGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:

(2) INFORMATION FOR SEQ ID NO: 141:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 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 pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:

(2) INFORMATION FOR SEQ ID NO: 142:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRAND~nN~.~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQu~ DESCRIPTION: SEQ ID NO: 142:

(2) lN~-O~ATION FOR SEQ ID NO: 143:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH
modified CA 02223003 l997-l2-0l W096/40717 PCT~S96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:
GGGAGACAAG AAUA~ACGCU CAACCGUAGC CUCCAGCGGA ACGCGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 144:
O (i) SEQuh~h CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQ~hN~h DESCRIPTION: SEQ ID NO: 144:
GGGAGACAAG AAUAAACGCU CAAUGCCGAG AGGAGGGCUG A~GAGGACGC 50 (2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:

(2) INFORMATION FOR SEQ ID NO: 146:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146:

(2) INFORMATION FOR SEQ ID NO: 147:

CA 02223003 l997-l2-0l (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQD~N-~ DESCRIPTION: SEQ ID NO: 147:

(2) INFORMATION FOR SEQ ID NO: 148:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148:
GGGAGACAAG AAUA~ACGCU CA~ACGUAGC CUCCAGCGGA AUGCGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 149:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149:
GGGAGACAAG AAUA~ACGCU CAAGCGGUCU GAUCGAGCCU CCGUGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
- (D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:

(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: .l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SE~u~ DESCRIPTION: SEQ ID NO: 151:
GGGAGACAAG AAUA~ACGCU CAAUGCCGAG AGGAGGGCUG AGGAGGACAC 50 (2) INFORMATION FOR SEQ ID NO: 152:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified (xi) SEQU~ DESCRIPTION: SEQ ID NO: 152:
GGGAGACAAG AAUAAACGCU CAACCGUAGC CUCCAGCGGA ~UGUGGAGGG 50 (2) INFORMATION FOR SEQ ID NO: 153:
(i) SEQUENCE CHARACTBRIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NH2 modified CA 02223003 l997-l2-0l W096/40717 PCT/U~G~ 537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:
GGGAGACAAG AAUAAACGCU CAAAAGGUGG ~uC~u~GAGG AAUGAGCUCG 50 (2) INFORMATION FOR SEQ ID NO: 154:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U'~ are 2'-NH2 modified (ix) FEAlUKE:
(D) OTHER INFORMATION: All C's are 2'-F modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154:

(2) INFORMATION FOR SEQ ID NO: 155:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U'8 are 2 '-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:
GGGAGACAAG AAUAAACGCU CAA~u~uuCC AUGGGGAGCC ACA W AACGG 50 (2) INFORMATION FOR SEQ ID NO: 156:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 ~ase pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: RNA
(iX) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH~ modified (ix) FEATURE:
(D) OTHER INFORMATION: All C'5 are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:
GGGAGACAAG AAUAAACGCU CAACUCGGGA GCCAGAGUAA CA~CGGCACU 50 CA 02223003 l997-l2-0l WO96/40717 PCT/U',G/~5J37 (2) INFORMATION FOR SEQ ID NO: 157:
(i) SEQu~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:
GGGAGACAAG AAUAAACGCU CA~AGAGCCG W W GGGGAC CCACAGUAAC 50 (2) INFORMATION FOR SEQ ID NO: 158:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C' 6 are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158:

(2) INFORMATION FOR SEQ ID NO: 159:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 82 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:
GGGAGACAAG AAUAAACGCU CA~AGUAACG U&GGGAGCCA CACGUAAUAC 50 CA 02223003 l997-l2-0l WO96/40717 PCT~US96/09537 (2) INFORMATION FOR SEQ ID NO: 160:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:

(2) INFORMATION FOR SEQ ID NO: 161:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C'~ are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:
GGGAGACAAG AAUA~ACGCU CAAGAUCCUG CGACGCCAGG GGUGGAUAGG 50 (2) INFORMATION FOR SEQ ID NO: 162:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U'~ are 2'-NHz modified (ix) FEATURE:
(D) OTHER INFORMATION: All C'~ are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162:

AAUAGGGAUG CC~uuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 163:
(i) SEQUENCE CHARACTERIZATION:

CA 02223003 l997-l2-0l W096/40717 PCT~S96/09537 (A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
~ (ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:
GGGAGACAAG AAUA~ACGCU CAAGGUACGA CCAAGGAAUG UGGGUGGAAG 50 (2) INFORMATION FOR SEQ ID NO: 164:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:
GGGAGACAAG AAUA~ACGCU CAACAACGCU GACCAUGGGA GGAAUGUGGG 50 (2) INFORMATION FOR SEQ ID NO: 165:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-E~ modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:
GGGAGACAAG AAUA~ACGCU CAACAGCCA~ GGG W GGAUA GGGGGUAGGG 50 (2) INFORMATION FOR SEQ ID NO: 166:
(i) SEQUENCE CHARACTERIZATION:
(A) ~ENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAl~KE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:

UGAGAGCAAC A~uuuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 167:
(i) SE~u~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D)- TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:
GGGAGACA~G AAUAAACGCU CAAAAGGUGG G W GAGGAGG AAAGUAGCGU 50 (2) INFORMATION FOR SEQ ID NO: 168:
(i) SEQUhN~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEAlu~E:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:

(2) INFORMATION FOR SEQ ID NO: 169:
(i) SE~u~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-MH2 modified /: ~ T -~ ~ TTD T:~
(D) OTHER INFORMATION: All C's are 2'-E' modified s~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169:

(2) INFORMATION FOR SEQ ID NO: 170:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-E7 modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170:
GGGAGACAAG AAUA~ACGCU QAGGGUGGA W GUGGAGGA AGUAGCG QG 50 GG W CCGUAA GCC W CGA Q GGAGGCU QC AA QGGC . 87 (2) INFORMATION FOR SEQ ID NO: 171:
(i) SE~u~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171:
GGGAGACAAG AAUAAACGCU CA~AGGAGCG C QUGAAGCA AAGGGAGGAU 50 (2) INFORMATION FOR SEQ ID NO: 172:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:

CA 02223003 l997-l2-0l WO96/40717 PCTrUS96/09537 (D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172: .

~u~u~uGGAG GA W CGACAG GAGGCUCACA ACAGGC 86 (2) INFORMATION FOR SEQ ID NO: 173:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U' 6 are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C'~ are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173:
GGGAGACAAG AAUAAACGCU CA~AGGGAGG A W GUGGAGG AAGGGAGUGG 50 AA~u~U~u~A GCC W CGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 174:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 174:

G~uu~u~GAG GAG W CGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 175:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (D) OTHER INFORMATION: All C's are 2'-F modified - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175:
GGGAGACAAG AAUAAACGCU CA~ACCUGAU AACCGCGGAG GGAGGAUAGA 50 (2) INFORMATION FOR SEQ ID NO: 176:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 176:
GGGAGACAAG AAUA~ACGCU CAAAGGCAGC CCCUCGACGA GA~AGGUGGG 50 (2) INFORMATION FOR SEQ ID NO: 177:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAlu~E:
(D) OTHER INFORMATION: All U's are 2'-~2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQu~ DESCRIPTION: SEQ ID NO: 17'1:
GGGAGA Q AG AAUAAACGCU QAC W ACGA QC QAAGGG i~GGA WGUGG 50 UGGAAUGGGG uC~uuC~A Q GGAGGCU QC AA QGGC 87 (2) INFORMATION FOR SEQ ID NO: 178:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 178:

CA 02223003 l997-l2-0l W096/40717 PCTrUS96/09537 (2) INFORMATION FOR SEQ ID NO: 179:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEAluKE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQu~N~: DESCRIPTION: SEQ ID NO: 179:

(2) INFORMATION FOR SEQ ID NO: 180:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 180:

(2) INFORMATION FOR SEQ ID NO: 181:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 181:

(2) INFORMATION FOR SEQ ID NO: 182:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-NHz modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 182:

(2) INFORMATION FOR SEQ ID NO: 183:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH, modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 183:

(2) INFORMATION FOR SEQ ID NO: 184:
(i) SEyu~:N~ CHARACTERIZATION:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATuKE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 184:

(2) INFORMATION FOR SEQ ID NO: 185:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All U's are 2'-NH2 modified CA 02223003 l997-l2-0l (ix) FEATURE:
(D) OTHER INFORMATION: All C's are 2'-F modi~ied (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 185:

(2) INFORMATION FOR SEQ ID NO: 186:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 186:
GGGAGACAAG AAUAAACGCU CA~NNNNNNN NNNNNNNNNN NNNNNNNNN~ 50 -NNNNNNNNNN NNN U U C~AcA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 187:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 187:

(2) INFORMATION FOR SEQ ID NO: 188:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 188:
GC~l~llvlG AGCCTCCTGT CGAA 24 (2) INFORMATION FOR SEQ ID NO: 189:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 base pairs (18) TYPE: nucleic acid (C) STRANDEDNESS: 5 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 189:

(2) INFORMATION FOR SEQ ID NO: 190:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 190:

(2) INFORMATION FOR SEQ ID NO: 191:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 191:

(2) INFORMATION FOR SEQ ID NO: 192:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) sTR~n~n~-ss single (D) TOPOLOGY: line~
(ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 192:

(2) INFORMATION FOR SEQ ID NO: 193:

CA 02223003 l997-l2-0l (i) SEQuhN~ CHARACTERIZATION:
(A) LENGTH: 85 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 193:
GGGAGACAAG AAUAAACGCU CAAACAUCGU AUAAu~u~uA AGAGCCUGGA 50 (2) INFORMATION FOR SEQ ID NO: 194:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SE~u~N~ DESCRIPTION: SEQ ID NO: 194:
GGGAGACAAG AAUAAACGCU CAAACAUCGU AUAAu~u~uC UAAGAGCCUG 50 (2) INFORMATION FOR SEQ ID NO: 195:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 195:
GGGAGACAAG AAUAAACGCU CAAAUCCCAA u~u~uAAGAG CCUGGAUAAG 50 (2) INFORMATION FOR SEQ ID NO: 196:
(i) SEQu~ CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear CA 02223003 l997-l2-0l W096/40717 PCT~S96/09537 (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 196:
GGGAGACAAG AAUAAACGCU CA~AUCCCAA UCUCUAAGAG CCUGGAUGAC 50 (2) INFORMATION FOR SEQ ID NO: 197:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l; ne~r (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 197:
GGGAGACAAG AAUA~ACGCU CA~AUCCCAA UCUCUAAGAG CCUGGAUGAG 50 (2) INFORMATION FOR SEQ ID NO: 198:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 198:
GGGAGACAAG AAUA~ACGCU CAACUGAGAU CUCUAAGAGC CUGGACUCAG 50 (2) INFORMATION FOR SEQ ID NO: 199:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l99:

(2) INFORMATION FOR SEQ ID NO: 200:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 200:
GGGAGACAAG AAUA~ACGCU CAACUGAGAU CUCUAAGAGC CUGGACUCAG 50 (2) INFORMATION FOR SEQ ID NO: 20l:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 201:
GGGAGACAAG AAUAAACGCU CAAu~u~uAU GAGCCUGGAU CGACGAACUC 50 UCUACGGGCU ~u~uuCGACA GGAGGCUCAC AACAGGC 87 (2) INFORMATION FOR SEQ ID NO: 202:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modified (xi) s~QU~ DESCRIPTION: SEQ ID NO: 202:

(2) INFORMATION FOR SEQ ID NO: 203:

WO96/40717 PCT~S96/09537 (i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 203:

A~uuuuAUCA CAG WW CGAC AGGAGGCUCA CAACAGGC 88 (2) INFORMATION FOR SEQ ID NO: 204:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 87 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(ix) FEATURE:
(D) OTHER INFORMATION: All pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 204:
GGGAGACAAG AAUAAACGCU CAACGUAAAA G W AUCGAAU ~u~u~u~AGC 50 (2) INFORMATION FOR SEQ ID NO: 205:
(i) SEQUENCE CHARACTERIZATION:
(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 pyrimidines are 2'-F
modi~ied (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 205:

CUACGA W GA GCG WW A W C uu~uuCGACA GGAGGCUCAC AACAGGC 97 (2) INFORMATION FOR SEQ ID NO: 206:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: RNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 206:
GGGAGCUCAG AAUAAACGCU CA~NNNNNNN NNNNNNNNN~ ~NNNNNNNN~ 50 NNNuuC~ACA UGAGGCCCGG AUCCGGC 77 (2) INFORMATION FOR SEQ ID NO: 207:
(i) SEQu~N~: CHARACTERIZATION:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 207:

(2) INFORMATION FOR SEQ ID NO: 208:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 208:

(2) INFORMATION FOR SEQ ID NO: 209:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 209:

(2) INFORMATION FOR SEQ ID NO: 2l0:
(i) S~Qu~NCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) S~QU~NCE DESCRIPTION: SEQ ID NO: 210:
GGGAGCTCAG AATAAACGCT CAAGCGCTTG ACCAlll~l AGGGTCGCCC 50 CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (2) INFORMATION FOR SEQ ID NO: 211:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: 6 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 211:

(2) INFORMATION FOR SEQ ID NO: 212:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOhOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 212:

ACGTTCGACA TGAGGCCCGG ATCCGGC . 77 (2) INFORMATION FOR SEQ ID NO: 213:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 213:

(2) INFORMATION FOR SEQ ID NO: 214:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l in~r (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 214:
GGGAGCTCAG AATA~ACGCT CAACCACTGG CTAGGAACTC GAGTACTGGG 50 CA 02223003 l997-l2-0l WO96/40717 PCT~US96/09537 (2) INFORMATION FOR SEQ ID NO: 215:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -~
(ii) MOLECULAR TYPE: DNA
(xi) SEQ~N~h DESCRIPTION: SEQ ID NO: 215:

(2) INFORMATION FOR SEQ ID NO: 216:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRAN~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 216:

(2) INFORMATION FOR SEQ ID NO: 217:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SÉQUENCE DESCRIPTION: SEQ ID NO: 217:

(2) INFORMATION FOR SEQ ID NO: 218:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 218:

(2) INFORMATION FOR SEQ ID NO: 219:
(i) SEQu~-~ CHARACTERIZATION:

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (A) LENGTH: 77 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 219:

(2) INFORMATION FOR SEQ ID NO: 220:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 220:

(2) INFORMATION FOR SEQ ID NO: 221:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRAND~N~.~S: ~ingle (D) TOPOLOGY: l; neAr (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 221:

(2) INFORMATION FOR SEQ ID NO: 222:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 222:
GGGAGCTCAG AATA~ACGCT CAACGTTGAA CGCTTGGTTT CATGTCCCTC 50 (2) INFORMATION FOR SEQ ID NO: 223:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid CA 02223003 l997-l2-0l (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 223: .

(2) INFORMATION FOR SEQ ID NO: 224:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 224:

(2) INFORMATION FOR SEQ ID NO: 225:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 225:

(2) INFORMATION FOR SEQ ID NO: 226:
(i) SE~ N~: CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 226:

(2) INFORMATION FOR SEQ ID NO: 227:
(i) SE~u~N~ CHARACTERIZATION:
(A) LENGTH: 77 ba~e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO96/40717 PCT~S96/09~37 (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 227:

CTGTTCGACA TGAGGCCCGG ATCCGGC . 77 (2) INFORMATION FOR SEQ ID NO: 228:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 228:

(2) INFORMATION FOR SEQ ID NO: 229:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 229:

(2) INFORMATION FOR SEQ ID NO: 230:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 230:
GGGAGCTCAG AATAAACGCT CAAGTCGGAT G~llllGCGC GTTTCCCGTT 50 (2) INFORMATION FOR SEQ ID NO: 231:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA

WO96140717 PCT~US96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 231:

(2) INFORMATION FOR SEQ ID NO: 232:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 232:

(2) INFORMATION FOR SEQ ID NO: 233:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 233:

(2) INFORMATION FOR SEQ ID NO: 234:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRA~ ~SS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 234:

(2) INFORMATION FOR SEQ ID NO: 235:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRAND~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 235:
GGGAGCTCAG AATAAACGCT CAAGTCGCTC GAl-C~ll-l~A l~lCC~llCG 50 CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (2) INFORMATION FOR SEQ ID NO: 236:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 74 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 236:

(2) INFORMATION FOR SEQ ID NO: 237:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 237:

CCTTCGACAT GAGGCCCGGA TCCGGC . 76 (2) INFORMATION FOR SEQ ID NO: 238:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 238:
GGGAGCTCAG AATA~ACGCT CAACGTCGAC GCACTGTGCC GCCTCACACA 50 (2) INFORMATION FOR SEQ ID NO: 239:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 ba6e pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 239:

CA 02223003 l997-l2-0l (2) INFORMATION FOR SEQ ID NO: 240:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~ DESCRIPTION: SEQ ID NO: 240:

(2) INFORMATION FOR SEQ ID NO: 241:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 241:

(2) INFORMATION FOR SEQ ID NO: 242:
(i) SEyu~b: CHARACTERIZATION:
(A) LENGTH: 77 base pair6 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 242:

(2) INFORMATION FOR SEQ ID NO: 243:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 243:
GGGAGCTCAG AATA~ACGCT CAACAGGTGG CACCGCCCTT CCAACACGGT 50 (2) INFORMATION FOR SEQ ID NO: 244:
(i) SEQUENCE CHARACTERIZATION:

WO96/40717 PCT~S96/09537 (A) LENGTH: 74 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 244:

(2) INFORMATION FOR SEQ ID NO: 245:
(i) SEQ~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: 1 inear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 245:

(2) INFORMATION FOR SEQ ID NO: 246:
(i) SEQuhN~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRAN~SS: 8 ingle (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 246:
GGGAGCTCAG AATAAACGCT CAACGCCGAG ACCCACCTCA ~AACACCGCT 50 (2) INFORMATION FOR SEQ ID NO: 247:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l inear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 247:

(2) INFORMATION FOR SEQ ID NO: 248:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid CA 02223003 l997-l2-0l W096/40717 PCTrUS96/09537 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 248: .

(2) INFORMATION FOR SEQ ID NO: 249:
~i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 249:

(2) INFORMATION FOR SEQ ID NO: 250:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 8 ingle (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 250:

(2) INFORMATION FOR SEQ ID NO: 251:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 251:

(2) INFORMATION FOR SEQ ID NO: 252:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 5 ingle (D) TOPOLOGY: linear WO96/40717 PcT/u~c~ J37 (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 252:

(2) INFORMATION FOR SEQ ID NO: 253:
(i) SEQUENCE CHARACT~RT~TION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 253:

(2) INFORMATION FOR SEQ ID NO: 254:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 254:

(2) INFORMATION FOR SEQ ID NO: 255:
(i) SEUu~N~ CHARACTERIZATION:
(A) LENGTH: 77 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQu~ DESCRIPTION: SEQ ID NO: 255:
GGGAGCTCAG AATAAACGCT CAATGTCGAT CGTGTCAAGG l'CCGTCCTAC 50 (2) INFORMATION FOR SEQ ID NO: 256:
(i) S~Qu~NCE CHARACTERIZATION:
(A) LENGTH: 71 base pairs ~B) TYPE: nucleic acid (C) STR~n~nN~S: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA

CA 02223003 l997-l2-0l WO96/40717 PCT~S96/09537 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 256:
GGGAGGACGA TGcG(~NNNN~ ~NNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 (2) INFORMATION FOR SEQ ID NO: 257:
(i) SEQUENCE CHARACTERIZATION:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: DNA
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 257:

(2) INFORMATION FOR SEQ ID NO: 258:
(i) SEQu~N~ CHARACTERIZATION:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l; ne~r (ii) MOLECULAR TYPE: DNA
(ix) FEATURE:
(D) OTHER INFORMATION: N at positions 1-4 is biotin (xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 258:

Claims (46)

WE CLAIM:

1. A method of identifying nucleic acid ligands to a cytokine, comprising:
a) contacting a candidate mixture of nucleic acids with said cytokine, wherein nucleic acids having an increased affinity to said cytokine relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; and 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 cytokine, whereby nucleic acid ligands of said cytokine 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 of nucleic acids is comprised of single stranded nucleic acids.

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

5. The method of claim 4 wherein said nucleic acids are modified nucleic acids.

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

7. The method of claim 5 wherein said nucleic acids are 2'-fluoro (2'-F) modified ribonucleic acids.

8. The method of claim 3 wherein said single stranded nucleic acids are deoxyribonucleic acids.

9. The method of claim 1 wherein said cytokine is selected from the group consisting of IFN-gamma IL-10, IL-4, TNF-alpha, and RANTES.

10. The method of claim 1 wherein said cytokine is IFN gamma

11. The method of claim 1 wherein said cytokine is IL-10.

12. The method of claim l wherein said cytokine is IL-4.

13. The method of claim 1 wherein said cytokine is TNF-alpha.

14. The method of claim 1 wherein said cytokine is RANTES.

15. A method for treating a cytokine-mediated disease comprising administering apharmaceutically effective amount of a nucleic acid ligand of a cytokine.

16. The method of claim 15 wherein said nucleic acid ligand of a cytokine is identified according to the method of claim 1.

17. The method of claim 16 wherein said cytokine is IFN-gamma

18. The method of claim 17 wherein said ligand is selected from one ofthe ligands of Tables 3 and 4 (SEQ ID NOS:7-73).

19. The method of claim 16 wherein said cytokine is IL-4.

20. The method of claim 17 wherein said ligand is selected from one of the ligands of Tables 7 and 8 (SEQ ID NOS: 100-185).

21. The method of claim 16 wherein said cytokine is IL-10.

22. The method of claim 21 wherein said ligand is selected from one of the ligands of Table 10 (SEQ ID NOS:189-205).

23. The method of claim 16 wherein said cytokine is TNF-alpha.

24. The method of claim 23 wherein said ligand is selected from one of the ligands of Table 12 (SEQ ID NOS:209-255).

25. A purified and isolated non-naturally occurring nucleic acid ligand to a cytokine.

26. The purified and isolated non-naturally occurring nucleic acid ligand of claim 25 wherein said nucleic acid ligand is single-stranded.

27. The purified and isolated non-naturally occurring nucleic acid ligand of claim 26 wherein said nucleic acid ligand is ribonucleic acid.

28. The purified and isolated non-naturally occurring nucleic acid ligand of claim 26 wherein said nucleic acid ligand is deoxyribonucleic acid.

29. A nucleic acid ligand to a cytokine identified according to the method comprising:

a) contacting a candidate mixture of nucleic acids with said cytokine, wherein nucleic acids having an increased affinity to said cytokine relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; and 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 cytokine, whereby nucleic acid ligands of said cytokine may be identified.

30. The purified and isolated non-naturally occurring ribonucleic acid ligand of claim 27, wherein said ligand is IFN-gamma.

31. The purified and isolated non-naturally occurring ribonucleic acid ligand toIFN-gamma of claim 30 wherein said ligand is selected from the group consisting of the sequences set forth in Tables 3 and 4 (SEQ ID NOS:7-73).

32. The purified and isolated non-naturally occurring ribonucleic acid ligand toIFN-gamma of claim 30 wherein said ligand is substantially homologous to and hassubstantially the same ability to bind IFN-gamma as a ligand selected from the group consisting of the sequences set forth in Tables 3 and 4 (SEQ ID NOS:7-73).

33. The purified and isolated non-naturally according ribonucleic acid ligand toIFN-gamma of claim 30 wherein said ligand has substantially the same structure and substantially the same ability to bind IFN-gamma as a ligand selected from the group consisting of the sequences set forth in Tables 3 and 4 (SEQ ID NOS:7-73).

34. The purified and isolated non-naturally occurring ribonucleic acid ligand ofclaim 27 wherein said ligand is to IL 4.

35. The purified and isolated non-naturally occurring ribonucleic acid ligand toIL-4 of claim 34 wherein said ligand is selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:79-185).

36. The purified and isolated non-naturally occurring ribonucleic acid ligand toIL-4 of claim 34 wherein said ligand is substantially homologous to and has substantially the same ability to bind IL-4 as a ligand selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:79-185).

37. The purified and isolated non-naturally occurring ribonucleic acid ligand to IL-4 of claim 34 wherein said ligand has substantially the same structure and substantially the same ability to bind IL-4 as a ligand selected from the group consisting of the sequences set forth in Tables 7 and 8 (SEQ ID NOS:79-185).

38. The purified and isolated non-naturally occurring ribonucleic acid ligand ofclaim 27 wherein said ligand is to IL-10.

39. The purified and isolated non-naturally occurring ribonucleic acid ligand to IL-10 of claim 38 wherein said ligand is selected from the group consisting of the sequences set forth in Table 10 (SEQ ID NOS:189-205).

40. The purified and isolated non-naturally occurring ribonucleic acid ligand to IL-10 of claim 38 wherein said ligand is substantially homologous to and has substantially the same ability to bind IL-10 as a ligand selected from the group consisting of the sequences set foreth in Table 10 (SEQ ID NOS:189-205).

41. The purified and isolated non-naturally occurring ribonucleic acid ligand to IL-10 of claim 38 wherein said ligand has substantially the same structure and substantially the same ability to bind IL-10 as a ligand selected from the group consisting of the sequences set forth in Table 10 (SEQ ID NOS:189-205).

42. The purified and isolated non-naturally occurring ribonucleic acid ligand ofclaim 27 wherein said ligand is to TNF-alpha.

43. The purified and isolated non-naturally occurring ribonucleic acid ligand toTNF-alpha of claim 42 wherein said ligand is selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:209-255).

44. The purified and isolated non-naturally occurring ribonucleic acid ligand toTNF-alpha of claim 42 wherein said ligand is substantially homologous to and hassubstantially the same ability to bind TNF-alpha as a ligand selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:209-255).

45. The purified and isolated non-naturally occurring ribonucleic acid ligand toTNF-alpha of claim 42 wherein said ligand has substantially the same structure and substantially the same ability to bind TNF-alpha as a ligand selected from the group consisting of the sequences set forth in Table 12 (SEQ ID NOS:189-205).

46. The purified and isolated non-naturally occurring nucleic acid ligand of claim 25 wherein said ligand is to RANTES.