HIGH AFFINITY TGFB NUCLEIC ACID LIGANDS AND INHIBITORS FIELD OF THE INVENTION Described herein are methods for identifying and preparing high-affinity nucleic acid ligands to TGFB. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential enrichment. This invention includes high affinity nucleic acid ligands of TGFB. Further disclosed are RNA ligands to TGFB1. Also included are oligonucleotides containing nucleotide derivatives chemically modified at the 2'-positions of pyrimidines. Additionally disclosed are RNA ligands to TGFB1 containing 2'-F-modifications. This invention also includes high affinity nucleic acid inhibitors of TGFB1. The oligonucleotides of the present invention are useful as pharmaceuticals or diagnostic agents.

BACKGROUND OF THE INVENTION The transforming growth factor-B (TGFB) polypeptides influence growth, differentiation, and gene expression in many cell types. The first polypeptide of this family that was characterized, TGFB1 has two identical 112 amino acid subunits which are covalently linked. TGFB1 is a highly conserved protein with only a single amino acid difference distinguishing humans from mice. There are two other members of the TGFB gene family that are expressed in mammals. TGFB2 is 71 % homologous to TGFß 1 (de Martin et al. (1987) EMBO J. 6: 3673-3677), whereas TGFB3 is 80% homologous to TGFBI (Derynck et al. (1988) EMBO J 7: 3737-3743). The structural characteristics of TGFB I as determined by nuclear magnetic resonance (Archer et al. (1993) Biochemistry 32: 1164-1171) agree with the crystal structure of TGFB2 (Daopin et al. (1992) Science 257: 369-374; Schlunegger and Grutter (1992) Nature 358: 430-434).

Even though the TGFB's have similar three dimensional structures, they are by no means physiologically equivalent. There are at least three different extracellular receptors, type I, II and III, involved in transmembrane signaling of TGFB to cells carrying the receptors. (For reviews, see Derynck (1994) TIBS 19: 548-553 and Massague (1990) Ann

Rev. Cell Biol. 6: 597-641). In order for TGFB2 to effectively interact with the type II TGFB receptor, the type III receptor must also be present (Derynck (1994) TIBS 19: 548-553).

Vascular endothelial cells lack the type III receptor. Instead endothelial cells express a structurally related protein called endoglin (Cheifetz et al. (1992) J. Biol. Chem. 267: 19027- 19030), which only binds TGFB1 and TGFB3 with high affinity. Thus, the relative potency of the TGFB's reflect the type of receptor expressed in a cell and organ system.

In addition to the regulation of the components in the multifactorial signaling pathway, the distribution of the synthesis of TGFß polypeptides also affects physiological function. The distribution of TGFB2 and TGFB3 is more limited (Derynck et al. (1988) EMBO J 7: 3737-3743) than TGFßl, e. g., TGFB3 is limited to tissues of mesenchymal origin, whereas TGFB1 is present in both tissues of mesenchymal and epithelial origin.

TGFB1 is a multifunctional cytokine critical for tissue repair. High concentrations of TGFB1 are delivered to the site of injury by platelet granules (Assoian and Sporn (1986) J.

Cell Biol. 102: 1217-1223). TGFB1 initiates a series of events that promote healing including chemotaxis of cells such as leukocytes, monocytes and fibroblasts, and regulation of growth factors and cytokines involved in angiogenesis, cell division associated with tissue repair and inflammatory responses. TGFB1 also stimulates the synthesis of extracellular matrix components (Roberts et al. (1986) Proc. Natl. Acad. Sci. USA 83: 4167- <BR> <BR> <BR> <BR> 4171; Sporn et al. (1983) Science 219: 1329-1330; Massague (1987) Cell 49: 437-438) and most importantly for understanding the pathophysiology of TGFßl, TGFB1 autoregulates its own synthesis (Kim et al. (1989) J. Biol. Chem. 264: 7041-7045).

A number of diseases have been associated with TGFB1 overproduction. Fibrotic diseases associated with TGFB1 overproduction can be divided into chronic conditions such as fibrosis of the kidney, lung and liver and more acute conditions such as dermal scarring and restenosis. Synthesis and secretion of TGFB1 by tumor cells can also lead to immune suppression such as seen in patients with aggressive brain or breast tumors (Arteaga et al.

(1993) J. Clin. Invest. 92: 2569-2576). The course of Leishmanial infection in mice is drastically altered by TGFB1 (Barral-Netto et al. (1992) Science 257: 545-547). TGFB1 exacerbated the disease, whereas TGFB1 antibodies halted the progression of the disease in genetically susceptible mice. Genetically resistant mice became susceptible to Leishmanial infection upon administration of TGFB1.

The profound effects of TGFB1 on extracellular matrix deposition have been reviewed (Rocco and Ziyadeh (1991) in Contemporary Issues in Nephrology v. 23, "Hormones, Autocoids and the Kidney,"ed. Jay Stein, Churchill Livingston, NY pp. 391- 410; Roberts et al. (1988) Rec. Prog. Hormone Res. 44: 157-197) and include the stimulation of the synthesis and the inhibition of degradation of extracellular matrix components. Since the structure and filtration properties of the glomerulus are largely determined by the extracellular matrix composition of the mesangium and glomerular membrane, it is not surprising that TGFB1 has profound effects on the kidney. The accumulation of mesangial matrix in proliferative glomerulonephritis (Border et al. (1990) Kidney Int. 37: 689-695) and diabetic nephropathy (Mauer et al. (1984) J. Clin. Invest. 74: 1143-1155) are clear and dominant pathological features of the diseases. TGFB levels are elevated in human diabetic glomerulosclerosis (advanced neuropathy) (Yamamoto et al. (1993) Proc. Natl. Acad. Sci.

USA 90: 1814-1818). TGFB1 is an important mediator in the genesis of renal fibrosis in a number of animal models (Phan et al. (1990) Kidney Int. 37: 426; Okuda et al. (1990) J.

Clin. Invest. 86: 453). Suppression of experimentally induced glomerulonephritis in rats has been demonstrated by antiserum against TGFB1 (Border et al. (1990) Nature 346: 371) and by an extracellular matrix protein, decorin, which can bind TGFßl (Border et al. (1992) Nature 360: 361-363).

Too much TGFB1 leads to dermal scar-tissue formation. Neutralizing TGFB1 antibodies injected into the margins of healing wounds in rats have been shown to inhibit scarring without interfering with the rate of wound healing or the tensile strength of the wound (Shah et al. (1992) Lancet 339: 213-214). At the same time there was reduced angiogenesis, reduced number of macrophages and monocytes in the wound, and a reduced amount of disorganized collagen fiber deposition in the scar tissue.

TGFB1 may be a factor in the progressive thickening of the arterial wall which results from the proliferation of smooth muscle cells and deposition of extracellular matrix in the artery after balloon angioplasty. The diameter of the restenosed artery may be reduced 90% by this thickening, and since most of the reduction in diameter is due to extracellular matrix rather than smooth muscle cell bodies, it may be possible to open these vessels to 50% simply by reducing extensive extracellular matrix deposition. In uninjured pig arteries transfected in vivo with a TGFB1 gene, TGFB1 gene expression was associated with both extracellular matrix synthesis and hyperplasia (Nabel et al. (1993) Proc. Natl.

Acad. Sci. USA 90: 10759-10763). The TGFB1 induced hyperplasia was not as extensive as that induced with PDGF-BB, but the extracellular matrix was more extensive with TGFB1 transfectants. No extracellular matrix deposition was associated with FGF-1 (a secreted form of FGF) induced hyperplasia in this gene transfer pig model (Nabel (1993) Nature 362: 844-846).

There are several types of cancer where TGFB1 produced by the tumor may be deleterious. MATLyLu rat cancer cells (Steiner and Barrack (1992) Mol. Endocrinol. 6: 15- <BR> <BR> <BR> <BR> 25) and MCF-7 human breast cancer cells (Arteaga et al. (1993) Cell Growth and Differ.

4: 193-201) became more tumorigenic and metastatic after transfection with a vector expressing the mouse TGFB1. In breast cancer, poor prognosis is associated with elevated TGFB (Dickson et al. (1987) Proc. Natl. Acad. Sci. USA 84: 837-841; Kasid eí al. (1987) <BR> <BR> <BR> <BR> Cancer Res. 47: 5733-5738; Daly et al. (1990) J. Cell Biochem. 43: 199-211; Barrett-Lee et al. (1990) Br. J Cancer 61: 612-617; King et al. (1989) J. Steroid Biochem. 34: 133-138; <BR> <BR> <BR> <BR> Welch et al. (1990) Proc. Natl. Acad. Sci. USA 87: 7678-7682; Walker et al. (1992) Eur. J.<BR> <BR> <BR> <BR> <BR> <BR> <P>Cancer 238: 641-644) and induction of TGFB1 by tamoxifen treatment (Butta et al. (1992) Cancer Res. 52: 4261-4264) has been associated with failure of tamoxifen treatment for <BR> <BR> <BR> <BR> breast cancer (Thompson etal. (1991) Br. J Cancer 63: 609-614). Anti TGFB1 antibodies<BR> <BR> <BR> <BR> <BR> <BR> inhibit the growth of MDA-231 human breast cancer cells in athymic mice (Arteaga et al.

(1993) J. Clin. Invest. 92: 2569-2576), a treatment which is correlated with an increase in spleen natural killer cell activity. CHO cells transfected with latent TGFB1 also showed decreased NK activity and increased tumor growth in nude mice (Wallick et al. (1990) J.

Exp. Med. 172: 1777-1784). Thus, TGFB1 secreted by breast tumors may cause an endocrine immune suppression.

High plasma concentrations of TGFB1 have been shown to indicate poor prognosis for advanced breast cancer patients (Anscher et al. (1993) N. Engl. J. Med. 328: 1592-1598).

Patients with high circulating TGFB before high dose chemotherapy and autologous bone marrow transplantation are at high risk for hepatic veno-occlusive disease (15-50% of all patients with a mortality rate up to 50%) and idiopathic interstitial pneumonitis (40-60% of all patients). The implication of these findings is 1) that elevated plasma levels of TGFB1 can be used to identify at risk patients and 2) that reduction of TGFB1 could decrease the morbidity and mortality of these common treatments for breast cancer patients.

A method for the in vitro evolution of nucleic acid molecules with high affinity

binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in United States Patent Application Serial No. 07/536,428, filed June 11,1990, entitled"Systematic Evolution of Ligands by Exponential Enrichment,"now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10,1991, entitled"Nucleic Acid Ligands,"now issued as United States Patent No. 5,475,096, United States Patent Application Serial No. 07/931,473, filed August 17,1992, entitled"Methods for Identifying Nucleic Acid Ligands,"now United States Patent No. 5,270,163 (see also W091/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.

The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity.

Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.

The basic SELEX method may be modified to achieve specific objectives. For example, United States Patent Application Serial No. 07/960,093, filed October 14,1992, entitled"Method for Selecting Nucleic Acids on the Basis of Structure,"now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. (See United States Patent No. 5,707,796). United States Patent Application Serial No. 08/123,935, filed September 17,1993, entitled"Photoselection of Nucleic Acid Ligands,"now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. (See United States Patent No. 5,763,177). United States Patent Application

Serial No. 08/134,028, filed October 7,1993, entitled"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,"abandoned in favor of United States Patent Application Serial No. 08/443,957, now United States Patent No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed"Counter-SELEX."United States Patent Application Serial No. 08/143,564, filed October 25,1993, entitled"Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,"abandoned in favor of United States Patent Application Serial No. 08/461,061, now United States Patent No. 5,567,588) and United States Patent Application Serial No. 08/792,075, filed January 31,1997, entitled "Flow Cell SELEX,"now United States Patent No. 5,861,254, describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. United States Patent Application Serial No.

07/964,624, filed October 21,1992, entitled"Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,"now United States Patent No. 5,496,938, describes methods for obtaining improved Nucleic Acid Ligands after the SELEX process has been performed. United States Patent Application Serial No. 08/400,440, filed March 8,1995, entitled"Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX,"now United States Patent No.

5,705,337, describes methods for covalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8,1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,"abandoned in favor of United States Patent Application Serial No. 08/430,709, now United States Patent No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5-and 2'-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2'-amino modifications. United States Patent Application Serial No.

08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe).

United States Patent Application Serial No. 08/264,029, filed June 22,1994, entitled

"Novel Method of Preparation of Known and Novel 2'Modified Nucleosides by Intramolecular Nucleophilic Displacement,"describes oligonucleotides containing various 2'-modified pyrimidines. International Publication No. WO 98/30720, published July 16, 1998, entitled"Bioconjugation of Oligonucleotides,"describes a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5'-position of the nucleic acid ligands.

The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,"now United States Patent No. 5,637,459 and United States Patent Application Serial No. 08/234,997, filed April 28,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,"now United States Patent No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties of oligonucleotides with the desirable properties of other molecules. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION The present invention includes methods of identifying and producing nucleic acid ligands to transforming growth factor beta (TGFB) and the nucleic acid ligands so identified and produced. For the purpose of this application, TGFB includes human TGFßl, TGFß2, TGFB3 and TGFB's that are substantially homologous thereto. By substantially homologous it is meant a degree of amino acid sequence identity of 70% or more. In particular, RNA sequences are provided that are capable of binding specifically to TGFB1. Specifically included in the invention are the RNA ligand sequences shown in Table 3 (SEQ ID NOS: 6- 143). Also included in this invention are RNA ligands of TGFB1 that inhibit the function of TGFB1.

Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to TGFB comprising the steps of (a) preparing a candidate mixture of nucleic acids, (b) contacting the candidate mixture of nucleic acids with TGFB,

(c) partitioning between members of said candidate mixture on the basis of affinity to TGFB, and (d) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to TGFB.

More specifically, the present invention includes the RNA ligands to TGFB identified according to the above-described method, including those ligands shown in Table 3 (SEQ ID NOS: 6-143). Also included are nucleic acid ligands to TGFB that are substantially homologous to any of the given ligands and that have substantially the same ability to bind TGFB and inhibit the function of TGFB. Further included in this invention are nucleic acid ligands to TGFB that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind TGFB and inhibit the function of TGFB.

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

BRIEF DESCRIPTION OF THE FIGURES Figures 1A and 1B show the binding curves of rounds 0 (o), 14 (A), 15 (a) and 16 (-) of the 40N pool (Fig. 1A) and rounds 0 (o), 14 (-), 15 (A) and 17 (-) of the 3 ON pool (Fig. 1B) presented as % RNA bound vs. concentration of TGFB 1.

Figure 2 shows the affinity sensorgram of random RNA (0), ligand 40-03 (o), ligand 40-60 (A) and polyclonal anti TGFB1 antibody (-) performed on TGFßl, expressed as response units vs. time.

Figures 3A-3C show sensorgrams obtained in a binding specificity analysis of TGFB1 performed on random RNA (Fig. 3A), ligand 40-03 (Fig. 3B) and ligand 40-60 (Fig.

3C) with various concentrations of TGFßl, expressed as response units vs. time. Figures 3D-3F show sensorgrams obtained in a binding specificity analysis of TGFB2 performed on random RNA (Fig. 3D), ligand 40-03 (Fig. 3E) and ligand 40-60 (Fig. 3F) with various concentrations of TGFß2, expressed as response units vs. time.

Figures 4A and 4B illustrate the results of the TGFB1 bioasay on mink lung epithelial cells (MLEC). Figures 4A and 4B show the inhibitory activity of rounds 11 (N) and 14 (a) of the 40N pool (Fig. 4A) and rounds 11 (a) and 14 () of the 3 ON pool (Fig. 4B) compared to random RNA (A). The results are expressed as 3H-thymidine incorporation as

net % of control vs. concentration of TGFß 1, where control is the amount of 3H-thymidine incorporation in the absence of TGFßl and RNA minus the amount of incorporation in the presence of TGFß 1 alone.

Figures 5A-5D illustrate the results of the TGFB1 bioassay on mink lung epithelial cells (MLEC). Figure SA is a TGFB1 titration curve presented as 3H-thymidine incorporation as a per cent of control vs. concentration of TGFBl. Figures 5B-5D illustrate the bioactivities of round 16 of the 40N pool (Fig. SB, (e)), ligand 40-03 (Fig. 5C, ()) and ligand 40-60 (Fig. 5D, ()) as compared to the bioactivities of a polyclonal anti-TGFß 1 antibody (o) and random RNA (), presented as 3H-thymidine incorporation as a per cent of control vs. concentration ofTGFBl.

Figure 6 shows the bioactivities of random RNA (), ligand 40-60 (A), ligand 40-03 (), a monoclonal antibody specific for TGFB2 and TGFB3 (o) and a pan-specific antibody specific for TGFßl, TGFß2 and TGFB3 (A), presented as 3H-thymidine incorporation as a per cent of control vs. concentration ofTGFBl.

Figure 7 is a proposed folding of the class 1 bioactive ligands. S 1, S2 and S3 designate stem 1, stem 2 and stem 3 of the proposed structure.

DETAILED DESCRIPTION OF THE INVENTION This application describes high-affinity nucleic acid ligands to TGFB identified through the method known as SELEX. SELEX is described in United States Patent Application Serial No. 07/536,428, entitled"Systematic Evolution of Ligands by EXponential Enrichment,"now abandoned, United States Patent Application Serial No.

07/714,131, filed June 10,1991, entitled"Nucleic Acid Ligands,"now United States Patent No. 5,475,096, United States Patent Application Serial No. 07/931,473, filed August 17, 1992, entitled"Methods for Identifying Nucleic Acid Ligands,"now United States Patent No. 5,270,163, (see also W091/19813). These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications. Certain terms used to described the invention herein are defined as follows.

"Nucleic Acid Ligand"as used herein is a non-naturally occurring nucleic acid having a desirable action on a target. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching

to the target as in a suicide inhibitor, and facilitating the reaction between the target and another molecule. In the preferred embodiment, the desirable action is specific binding to a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule. Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids.

"Candidate Mixture"is a mixture of nucleic acids of differing sequence from which to select a desired ligand. The source of a candidate mixture can be from naturally- occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques. In a preferred embodiment, each nucleic acid has fixed sequences surrounding a randomized region to facilitate the amplification process.

"Nucleic Acid"means either DNA, RNA, single-stranded or double-stranded and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3'and 5'modifications such as capping.

"SELEX"methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with

amplification of those selected nucleic acids. Iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEX methodology is employed to obtain nucleic acid ligands to TGFB. The SELEX methodology is described in the SELEX Patent Applications.

"Target"means any compound or molecule of interest for which a ligand is desired.

A target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. In this application, the target is a TGFB, preferably TGFB1.

In its most basic form, the SELEX process may be defined by the following series of steps.

1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (i. e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the target, or (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized (i. e., the probability of finding a base at any position being one in four) or only partially randomized (e. g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).

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

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the

partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.

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

5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer 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 candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.

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

The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in United States Patent Application No. 08/434,465, filed May 4,1995, entitled"Nucleic Acid Ligand Complexes."VEGF nucleic acid ligands that are associated with a lipophilic compound, such as diacyl glycerol or dialkyl glycerol, in a diagnostic or therapeutic complex are described in United States Patent Application Serial No.

08/739,109, filed October 25,1996, entitled"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes,"now United States Patent No. 5,859,228. VEGF nucleic acid ligands that are associated with a lipophilic compound, such as a glycerol lipid, or a non- immunogenic, high molecular weight compound, such as polyalkylene glycol, are further described in United States Patent Application Serial No. 08/897,351, filed July 21,1997, entitled"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes." VEGF nucleic acid ligands that are associated with a non-immunogenic, high molecular weight compound or lipophilic compound are also further described in WO 98/18480, published May 7,1998, entitled"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid

Ligand Complexes."Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.

Certain embodiments of the present invention provide a complex comprising one or more nucleic acid ligands to TGFB covalently linked with a non-immunogenic, high molecular weight compound or lipophilic compound. A complex as used herein describes the molecular entity formed by the covalent linking of the nucleic acid ligand of TGFB to a non- immunogenic, high molecular weight compound. A non-immunogenic, high molecular weight compound is a compound between approximately 100 Da to 1,000,000 Da, more preferably approximately 1000 Da to 500,000 Da, and most preferably approximately 1000 Da to 200,000 Da, that typically does not generate an immunogenic response. For the purposes of this invention, an immunogenic response is one that causes the organism to make antibody proteins. In a preferred embodiment of the invention, the non-immunogenic, high molecular weight compound is a polyalkylene glycol. In the most preferred embodiment, the polyalkylene glycol is polyethylene glycol (PEG). More preferably, the PEG has a molecular weight of about 10-80K. Most preferably, the PEG has a molecular weight of about 20-45K.

In certain embodiments of the invention, the non-immunogenic, high molecular weight compound can also be a nucleic acid ligand.

Another embodiment of the invention is directed to complexes comprised of a nucleic acid ligand to TGFB and a lipophilic compound. Lipophilic compounds are compounds that have the propensity to associate with or partition into lipids and/or other materials or phases with low dielectric constants, including structures that are comprised substantially of lipophilic components. Lipophilic compounds include lipids as well as non-lipid containing compounds that have the propensity to associate with lipids (and/or other materials or phases with low dielectric constants). Cholesterol, phospholipid and glycerol lipids, such as dialkylglycerol, diacylglycerol, and glycerol amide lipids are further examples of lipophilic compounds. In a preferred embodiment, the lipophilic compound is a glycerol lipid.

The non-immunogenic, high molecular weight compound or lipophilic compound may be covalently bound to a variety of positions on the nucleic acid ligand to TGFB, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5'or 3'terminus of the nucleic acid ligand to TGFB. In embodiments where the lipophilic

compound is a glycerol lipid, or the non-immunogenic, high molecular weight compound is polyalkylene glycol or polyethylene glycol, preferably the non-immunogenic, high molecular weight compound is bonded to the 5'or 3'hydroxyl of the phosphate group thereof. In the most preferred embodiment, the lipophilic compound or non-immunogenic, high molecular weight compound is bonded to the 5'hydroxyl of the phosphate group of the nucleic acid ligand. Attachment of the non-immunogenic, high molecular weight compound or lipophilic compound to the nucleic acid ligand of TGFß can be done directly or with the utilization of linkers or spacers.

A linker is a molecular entity that connects two or more molecular entities through covalent bonds or non-covalent interactions, and can allow spatial separation of the molecular entities in a manner that preserves the functional properties of one or more of the molecular entities. A linker can also be referred to as a spacer.

The complex comprising a nucleic acid ligand to TGFB and a non-immunogenic, high molecular weight compound or lipophilic compound can be further associated with a lipid construct. Lipid constructs are structures containing lipids, phospholipids, or derivatives thereof comprising a variety of different structural arrangements which lipids are known to adopt in aqueous suspension. These structures include, but are not limited to, lipid bilayer vesicles, micelles, liposomes, emulsions, lipid ribbons or sheets, and may be complexed with a variety of drugs and components which are known to be pharmaceutically acceptable. In the preferred embodiment, the lipid construct is a liposome. The preferred liposome is unilamellar and has a relative size less than 200 nm. Common additional components in lipid constructs include cholesterol and alpha-tocopherol, among others. The lipid constructs may be used alone or in any combination which one skilled in the art would appreciate to provide the characteristics desired for a particular application. In addition, the technical aspects of lipid constructs and liposome formation are well known in the art and any of the methods commonly practiced in the field may be used for the present invention.

The SELEX method further comprises identifying bioconjugates to a target.

Copending International Publication No. WO 98/30720, published July 6,1998, entitled "Bioconjugation of Oligonucleotides,"describes a method for enzymatically synthesizing bioconjugates comprising RNA derivatized exclusively at the 5'-position with a molecular entity, and a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5'-position of the nucleic acid

ligands. A bioconjugate as used herein refers to any oligonucleotide which has been derivatized with another molecular entity. In the preferred embodiment, the molecular entity is a macromolecule. As used herein, a macromolecule refers to a large organic molecule. Examples of macromolecules include, but are not limited to nucleic acids, oligonucleotides, proteins, peptides, carbohydrates, polysaccharides, glycoproteins, lipophilic compounds, such as cholesterol, phospholipids, diacyl glycerols and dialkyl glycerols, hormones, drugs, non-immunogenic high molecular weight compounds, fluorescent, chemiluminescent and bioluminescent marker compounds, antibodies and biotin, etc. without limitation. In certain embodiments, the molecular entity may provide certain desirable characteristics to the nucleic acid ligand, such as increasing RNA hydrophobicity and enhancing binding, membrane partitioning and/or permeability.

Additionally, reporter molecules, such as biotin, fluorescein or peptidyl metal chelates for incorporation of diagnostic radionuclides may be added, thus providing a bioconjugate which may be used as a diagnostic agent.

Certain embodiments of the present invention provide bioconjugates to TGFB comprising RNA derivatized exclusively at the 5'-position with a molecular entity obtained by the enzymatic method described in WO 98/30720. Other embodiments of the present invention provide bioconjugates to TGFB comprising a nucleic acid ligand covalently bonded to a macromolecule, obtained from a candidate mixture of bioconjugates, obtained by the method described in WO 98/30720.

The methods described herein and the nucleic acid ligands identified by such methods are useful for both therapeutic and diagnostic purposes. Therapeutic uses include the treatment or prevention of diseases or medical conditions in human patients.

Therapeutic uses may also include veterinary applications.

Diagnostic utilization may include both in vivo or in vitro diagnostic applications.

The SELEX method generally, and the specific adaptations of the SELEX method taught and claimed herein specifically, are particularly suited for diagnostic applications. SELEX identifies nucleic acid ligands that are able to bind targets with high affinity and with surprising specificity. These characteristics are, of course, the desired properties one skilled in the art would seek in a diagnostic ligand.

The nucleic acid ligands of the present invention may be routinely adapted for diagnostic purposes according to any number of techniques employed by those skilled in the

art or by the methods described in WO 98/30720, supra. Diagnostic agents need only be able to allow the user to identify the presence of a given target at a particular locale or concentration. Simply the ability to form binding pairs with the target may be sufficient to trigger a positive signal for diagnostic purposes. Those skilled in the art would also be able to adapt any nucleic acid ligand by procedures known in the art to incorporate a labeling tag in order to track the presence of such ligand. Such a tag could be used in a number of diagnostic procedures. The nucleic acid ligands to TGFB described herein may specifically be used for identification of the TGFB protein.

SELEX provides high affinity ligands of a target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to the specific target of TGFB1. In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligands to TGFB1 are described.

In order to produce nucleic acids desirable for use as a pharmaceutical, it is preferred that the nucleic acid ligand (1) binds to the target in a manner capable of achieving the desired effect on the target; (2) be as small as possible to obtain the desired effect; (3) be as stable as possible; and (4) be a specific ligand to the chosen target. In most situations, it is preferred that the nucleic acid ligand have the highest possible affinity to the target.

In the present invention, SELEX experiments were performed in order to identify RNA ligands with specific high affinity for TGFB1 from degenerate libraries containing 20, 30 or 40 random positions (20N7 (SEQ ID NO: 1), 30N7 (SEQ ID NO: 2) or 40N7 (SEQ ID NO: 3)) (Table 1). This invention includes the specific RNA ligands to TGFB1 shown in Table 3 (SEQ ID NOS: 6-143), identified by the methods described in Examples 1 and 2.

This invention further includes RNA ligands to TGFB1 which inhibit TGFB1 function, presumably by inhibiting the interaction of TGFB1 with its receptor. The scope of the ligands covered by this invention extends to all nucleic acid ligands of TGFB, modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to the ligands shown in Table 3 (SEQ ID NOS: 6-143). By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%, and even more preferably in excess of 90%, 95% or 99%. The percentage of homology as described herein is calculated as the percentage of nucleotides found in the smaller of the two sequences

which align with identical nucleotide residues in the sequence being compared when 1 gap in a length of 10 nucleotides may be introduced to assist in that alignment. A review of the sequence homologies of the ligands of TGFß shown in Tables 3 (SEQ ID NOS: 6-143) shows that some sequences with little or no primary homology may have substantially the same ability to bind TGFB. For this reason, this invention also includes nucleic acid ligands that have substantially the same structure and ability to bind TGFB as the nucleic acid ligands shown in Table 3 (SEQ ID NOS: 6-143). Substantially the same ability to bind TGFB means that the affinity is within one or two orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence--substantially homologous to those specifically described herein--has substantially the same ability to bind TGFB.

This invention also includes nucleic acid ligands that have substantially the same postulated structure or structural motifs. Substantially the same structure or structural motifs can be postulated by sequence alignment using the Zukerfold program (see Zuker (1989) Science 244: 48-52) as would be known in the art, other computer programs can be used for predicting secondary structure and structural motifs. Substantially the same structure or structural motif of nucleic acid ligands in solution or as a bound structure can also be postulated using NMR or other techniques as would be known in the art.

One potential problem encountered in the therapeutic, prophylactic, and in vivo diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e. g., United States Patent Application Serial No. 08/117,991, filed September 8,1993, entitled"High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,"abandoned in favor of United States Patent Application Serial No. 08/430,709, now issued as United States Patent No. 5,660,985 and United States Patent Application Serial No. 08/434,465, filed May 4,1995, entitled"Nucleic Acid Ligand Complexes,"which are specifically incorporated herein by reference. Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding,

electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3'and 5'modifications such as capping.

Where the nucleic acid ligands are derived by the SELEX method, the modifications can be pre-or post-SELEX modifications. Pre-SELEX modifications yield nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability. Post- SELEX modifications made to 2'-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand. The preferred modifications of the nucleic acid ligands of the subject invention are 5'and 3' phosphorothioate capping and/or 3'-3'inverted phosphodiester linkage at the 3'end. In one preferred embodiment, the preferred modification of the nucleic acid ligand is a 3'-3'inverted phosphodiester linkage at the 3'end. Additional 2'-fluoro (2'-F) and/or 2'-amino (2'-NH2) and/or 2'-O methyl (2'-OMe) modification of some or all of the nucleotides is preferred.

Described herein are nucleic acid ligands that were 2'-F modified and incorporated into the SELEX process. Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEX (modification of previously identified unmodified ligands) or by incorporation into the SELEX process.

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

Therapeutic compositions of the nucleic acid ligands may be administered parenterally by injection, although other effective administration forms, such as intraarticular injection, inhalant mists, orally active formulations, transdermal iontophoresis or suppositories, are also envisioned. One preferred carrier is physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers may also be used. In one preferred embodiment, it is envisioned that the carrier and the ligand constitute a

physiologically-compatible, slow release formulation. The primary solvent in such a carrier may be either aqueous or non-aqueous in nature. In addition, the carrier may contain other pharmacologically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.

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

Once the therapeutic composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder.

Such formulations may be stored either in a ready to use form or requiring reconstitution immediately prior to administration. The manner of administering formulations containing nucleic acid ligands for systemic delivery may be via subcutaneous, intramuscular, intravenous, intranasal or vaginal or rectal suppository.

The following Examples are provided to explain and illustrate the present invention and are not intended to be limiting of the invention. Example 1 describes the various materials and experimental procedures used in Example 2. Example 2 describes a representative method for identifying RNA ligands by the SELEX method which bind TGFB1. Example 3 describes the affinities the ligands have for TGFB1. Example 4 describes the specificity of ligands to hTGFßl. Example 5 describes the inhibition of TGFB1 bioactivity with several ligands. Example 6 summarizes the results of the data from Examples 2-5. Example 7 describes the proposed secondary structure ofbioactive TGFB1 ligands.

EXAMPLES Example 1. Experimental Procedures a) Materials Recombinant human Transforming Growth Factor Beta 1 (hTGFßl) was purchased from R&D Systems (Minneapolis, MN). Mink Lung Epithelial Cells (MLEC) were obtained from American Type Culture Collection (MV 1 Lu ATCC No. CCL 64). T7 RNA polymerase, 2'-F-modified CTP and UTP were prepared in house. DNA oligonucleotides

were obtained from Operon Technologies, Inc. (Alameda, CA). All other reagents and chemicals were from commercial sources. b) SELEX The SELEX process has been described in detail in United States Patent No.

5,270,163 (see also Tuerk and Gold (1990) Science 249: 505-510). The DNA templates contained either 40 (SEQ ID NO: 1), 30 (SEQ ID NO: 2) or 20 (SEQ ID NO: 3) random nucleotides, flanked by 5'and 3'constant regions for primer annealing sites for PCR and cDNA synthesis (Table 1). The starting pool of single stranded DNA molecules were converted to double stranded DNA by primer extension reactions with the klenow fragment of DNA polymerase. RNA pools were prepared by transcription and were gel purified before use. Transcription reactions were done with about 5 p1M DNA template, 5 units/pL T7 RNA polymerase, 40 mM Tris-HCl (pH 8), 12 mM MgCl2,5 mM DTT, 1 mM spermidine, 0.002% Triton X-100,4% PEG 8000,2-4 mM each 2'-OH ATP, 2'-OH GTP, 2'- F CTP, 2'-F UTP, and 0.25 uM a-"P-2'-OH ATP (800 Ci/mmole). At later rounds, RNA pools were prefiltered and/or preadsorbed with multiple layers of the same nitrocellulose filter type used in the SELEX process in order to reduce the frequency of molecules selected for nitrocellulose binding. To prepare binding reactions, the RNA molecules were incubated with recombinant HTGFBI in Dulbecco's Phosphate-Buffered Saline (DPBS) (Life Technologies, Gaithersburg, MD, Cat. No 21600-010) containing 0.01% human serum albumin and 1. 0 mM MgCl2. Following incubation at 37°C (10 minutes to 10 hours) the protein-RNA complexes were partitioned from unbound RNA by capture on nitrocellulose.

Nitrocellulose filter bound RNA was recovered by phenol/urea extraction. The partitioned RNA was reverse transcribed into cDNA by AMV reverse transcriptase at 48°C for 60 minutes in 50 mM Tris-HCl pH 8.3,60 mM NaCl, 6 mM Mg (OAc) 2,10 mM DTT, 50 pmol DNA 3'primer 3G7 (SEQ ID NO: 5; Table 1), 0.4 mM each of dATP, dCTP, dGTP, and dTTP, and 1 unit/nL AMV RT. The cDNA was PCR amplified and used to initiate the next SELEX cycle. PCR conditions were 2 RM each 3G7 (SEQ ID NO: 5) and 5G7 (SEQ ID NO: 4) primers (Table 1), 50 mM KCI, 10 mM Tris-HCI, pH 9,0.1% Triton X-100,3 mM MgCl2,0.5 mM of each dATP, dCTP, dGTP, and dTTP, and 0.1 units/pL Taq DNA polymerase.

c) NitrocelluloseFilterPartitioning To partition the protein-RNA complexes away from uncomplexed RNA, the binding reactions were filtered through nitrocellulose/cellulose acetate mixed matrix, 0.45 um pore size filter disks, type HA, (Millipore, Co., Bedford, MA). For filtration, the filters were placed onto a vacuum manifold and wetted by aspirating with 5 mL of DPBS. The binding reactions were aspirated through the filters, washed with 5 mL of DPBS + MgCl2 and counted in a scintillation counter (Beckmann). At later rounds, nitrocellulose filters were preblocked with 2 mL of DPBS + 1 mM MgCl2 + 0. 01 % BSA, and wash volumes were increased to 25 mL in order to reduce background binding to nitrocellulose. At later rounds in the SELEX process, 10 mL washes with 0.5 M urea were introduced to remove RNA that binds to nitrocellulose.

Nitrocellulose partitioning was also used for determining the equilibrium dissociation constants of RNA ligands to hTGFBl. Binding curves obtained by nitrocellulose filtration indicated that RNA pools and some RNA ligands bind monophasically while others bind biphasically. Biphasic binding can be described as the binding of two affinity species derived from the same ligand sequence that can fold into alternate structures which are kinetically trapped and are not in equilibrium.

To obtain the equilibrium dissociation constants of RNA ligands to TGFB1, the binding reaction: where R=RNA, P=Protein and KD=dissociation constant is converted into an equation for the fraction of RNA bound at equilibrium: q= (f/2RT) (PT+RT+ KD- ( (PT +RT+KD) 2-4PTRT)'iz) where q=fraction of RNA bound, PT=total protein concentration, RT=total RNA concentration and f=retention efficiency of RNA-protein complexes. The average retention efficiency for RNA-hTGFßl complexes on nitrocellulose filters is 0.4-0.8.

Biphasic binding data were evaluated using the equation: RT+KD1+KD2-[(PT+X1R1+KD1)2-4PTX1RT]1/2-[(PT+X1RT+KD2)2-4PTX2 RT]1/2q=2PT+ where X, and X2 are the mole fractions of the affinity species R, and R2 and KDI and KD2 are the corresponding dissociation constants.

The K. D's were determined by least square fitting of the data points using the software Kaleidagraph (Synergy Software, Reading, PA). d) Cloning and Sequencing RNA recovered from the filters of the final round of the SELEX process was reverse transcribed and PCR amplified as in previous rounds. The PCR products were purified by PAG electrophoresis and cloned into the Srfl restriction site of PCR-Script Direct SK (+) plasmid using the pCR-Script Amp SK (+) cloning kit (STRATAGENE CLONING SYSTEMS, La Jolla, CA). About 180 clones were sequenced with ABI Prism sequencing kit (Applied Biosystems, Perkin-Elmer, CT). e) Analysis of nucleic acid ligand binding by BIAcore Biotinylated TGFB1 (catalog No. NFTGO, R&D Systems, Minneapolis, MN) was coupled onto an SA5 streptavidin BIAcore chip (BIAcore, Inc., Piscataway, NJ) by injecting biotinylated TGF131 solution as prepared per manufacturers instructions at 5 pL/min to achieve loadings of 436,133 and 57 response units (RU) in flow cells 1,2 and 3, respectively. Flow cell 4 was kept blank for control and background subtractions. To measure binding activities, RNA ligands and antiserum (pan-specific anti-TGFßl total rabbit IgG, catalog No. AB-100-NA, R&D Systems, Minneapolis, MN) were injected at various concentrations in HBSMC-HSA (Hepes buffered saline pH 7.5,1 mM MgCl2,1 mM CaCl2,0.01% human serum albumin) at 20 pL/min. Injections allowed about 3 minute association and 3 minute dissociation cycles. Data were plotted and analyzed by Bianalysis software (BIAcore, Inc., Piscataway, NJ). fi Analysis of nucleic acid ligand specificity by BIAcore Biotinylated 2'-fluoro-pyrimidine RNA nucleic acid ligands were transcribed in the presence of 5'-biotin-modified guanosine monophosphate (5'-biotin-GAP) as described in copending International Publication No. WO 98/30720, published July 6,1998, the contents of which are incorporated herein by reference. Typical reactions were 1 mL in volume containing standard T7 RNA polymerase, 40 mM Tris-HCl (pH 8), 12 mM MgCl2, 5mM DTT, 1 mM spermidine, 0.002% Triton X-100,4% PEG 8000, with 3 mM each 2'-F-CTP and 2'-F-UTP, and 1 mM each ATP and GTP and 5 mM 5'-biotin GAP. Following overnight incubation at 37°C, transcripts were purified by gel electrophoresis and ethanol precipitation.

To prepare an analysis chip, three RNA species were used and were injected in HBSMC-HSA at 5 zL/min. Flow-cells 1,2 and 3 were loaded with 535,536 and 563 RU of random 40N7 library, TGFB1 ligand 40-03 (SEQ ID NO: 84), and TGFB1 ligand 40-60 (SEQ ID NO: 128), respectively. Thus, for stoichiometric binding of RNA to TGFB1 or TGFB2, one would expect a maximum of approximately 500 RU's, since TGFB1 and TGFB2 have the same mass as the RNA. Flow cell 4 was kept blank for control and background subtractions. The analysis chip was exposed to various concentrations of TGFB1 and TGFB2 at 20 uL/min. in HBSMC-HSA. Data were plotted and analyzed by Bianalysis software (BIAcore, Inc., Piscataway, NJ). g) Inhibition of TGFJYI mediated growth suppression of mink lung epithelial cells (MLEC) To determine the bioactivity of RNA pools and individual ligands, a growth assay was used in which TGFB I antagonists cause reversal of TGFB1 growth suppression of mink lung epithelial cells. In this assay, MLEC were treated with various concentrations of random RNA, individual ligands, antibodies such as polyclonal anti-TGFßl antibody (pan- specific anti-TGFßl total rabbint IgG, catalog No. AB-100-NA, R&D Systems, Minneapolis, MN), monoclonal mouse anti-TGFB2/TGFB3 antibody (Genzyme Corp., Cambridge MA, catalog No. 1836-01) and monoclonal mouse anti-TGFBl/TGFB2/ hTGFB3 antibody (Genzyme Corp., Cambridge, MA, catalog No. 1835-01) in serum-free 48 hr-3T3-conditioned medium (CM).

Cells were plated at 105/mL in 96-well plates in MEM, 10 mM HEPES and 0.2% FBS. Following 4 hours of incubation at 37°C, when cells appeared to attach to the well surface, TGFB1 was added at 2 pM with or without TGFB1 ligands that ranged from 0.1 nM to 1 pM. In a second assay performed in order to determine cross-species reactivity, rather than using hTGFß, a conditioned serum-free medium (CM) was used. CM was conditioned by culturing it in murine 3T3 fibroblast for 48 hours. Before use, this conditioned medium was heat treated at 80°C for 10 minutes to activate the 3T3 cell derived TGFB and then it was diluted to 50% and supplemented with 0.2% murine serum. In each assay, hTGFßl (or CM) was diluted appropriately in MEM and FBS (0.2% or murine serum) and the ligands were diluted in MEM. TGF131 (or CM) and ligand dilutions at 10X the final concentration were premixed at equal volumes and then were added to the cells. Following addition of the TGFB1 (or CM)-ligand mixture, the cells were incubated for 16-18 hours prior to addition

of 3H-thymidine at 0.25 pCi per well and continued incubation for 7-8 additional hours.

After incubation, the cells were washed and harvested with SKATRON filtering units and 3H-thymidine incorporation in cellular DNA was quantitated by scintillation counting in Ecoscint. Data were plotted and analyzed as described in Park et al. (1990) J. Exp. Med.

171: 1073) and Dower et al. (1984) J. Immunol. 132: 751). K ; values were determined from inhibition IC ; o values according to the equation Kj=ICso/ (l+ ( [T]/K), where [T] is the concentration in molar of TGFß 1 present in the assay and KdT is the concentration of TGFßl causing 50% inhibition of MLEC proliferation as determined by TGFB1 titration experiments.

Example 2. RNA ligands to hTGF131 a) TGFßl SELEX Three parallel SELEX processes were performed with 2'-F pyrimidine modified RNA randomized at 40,30 and 20 contiguous positions. The conditions for the SELEX process and results for each round are summarized in Table 2. The first round was done under two different conditions where RNA to protein ratios were 10: 1 and 50: 1. Each condition included a pool of 1.2xl 0'5 (2000 pmoles) 2'-F pyrimidine modified RNA molecules. Resulting round 1 pools were mixed (at the transcription level) in equal portions for round 2. Random 2'-F pyrimidine modified RNA bound to HTGFB I with an approximate KD of-10 nM. The rounds of the SELEX process were continued until no further improvement in KD was observed. Figures 1A and 1B show binding curves of rounds 0,14,15L and 16L of the 40N pool (Fig. 1A) and rounds 0,14,15 and 17 of the 3 ON pool (Fig. 1B). The 40N pools showed the best affinity improvement followed by the 30N pool. The 20N pool showed no significant improvement after 12 rounds of SELEX. The RNA pools from the final rounds (round 16,17 and 12 for the 40N, 30N and 20N, respectively) were reverse transcribed, PCR amplified and cloned as previously described (Pagratis et al. (1997) Nature Biotechnology 15: 68-73). The 20N pool was cloned and sequenced as a control. b) RNA sequences The sequences of 64,48, and 40 clones from the 40N, 30N and 20N final evolved pools, respectively, were determined and are summarized in Table 3 (SEQ ID NOS: 6-143) in standard single letter code (Cornish-Bowden (1985) Nucleic Acid Res. 13: 3021-3030).

Ligand designations in Table 3 include the size of the contributing random region followed by the ligand ID number. Ligands appearing more than once are designated with multiple ID numbers corresponding to their frequency. Ligands differing by one base are considered PCR derived variants of the same original molecule. Computer assisted global and local alignments suggest alignments and family assignments as shown in Table 4. There are 9 proposed families of which the first three include only 40N ligands. The remaining families contain clones derived from all three pools. However, it is clear from sequence lengths that cross contamination of the three pools had occurred. The possibility of cross contamination was minimized by electrophoretic size fractionation of RNA at each round, and PCR products prior to cloning.

Example 3. Binding Affinities of hTGF131 Ligands The dissociation constants of the hTGFB1 ligands were determined by nitrocellulose filter binding and are listed in Table 4. The majority of ligands bind hTGFB1 biphasically.

Under conditions of protein excess, biphasic binding suggests that ligands can exist as two affinity species (presumably isoconformers) that are not in equilibrium, i. e. isoconformers that are kinetically trapped. The best identified ligands, 40-03 (SEQ ID NO: 84) and 40-60 (SEQ ID NO: 128) bind biphasically with the high and low affinity dissociation constant of ligand 40-03 at about 0.3 pM and 4.6 nM, respectively. There are observed variabilities in the KD determinations for individual clones and random RNA, however, the high affinity species of ligands 40-03 and 40-60 always show about > 104 better affinity than random RNA in any given experiment. A significant difference between random RNA and ligands 40-03 and 40-60 was also observed by BIAcore analysis. In the BIAcore analysis, biotinylated TGF131 was coupled to a BIAcore chip and exposed to various concentrations of random RNA, ligand 40-03 and ligand 40-60. Also in this experiment the binding activities of ligands 40-03 and 40-60 were compared with the binding activity of an anti-TGFß 1 polyclonal antibody (catalog No. AB-100-NA, R&D Systems, Minneapolis, MN). Figure 2 shows the ligand binding of the random RNA, ligands 40-03 and 40-60, and of the anti- TGFB I antibody. From these Biacore data the determined dissociation rate constant (k off) for ligand 40-03, ligand 40-60 and anti-TGFßl were about oxo 04 and 4.4x10-5, respectively. Therefore, ligands 40-03 and 40-60 show binding properties similar to the

control antibody with the off rate of 40-03 being about 6 fold faster than the off rate of the anti-TGFBl.

Example 4. Specificity of RNA Ligands to hTGFßl The specificity of ligands 40-03 (SEQ ID NO: 84) and 40-60 (SEQ ID NO: 128) to TGFB1 was tested by comparing their dissociation constants with the closely related protein TGFB2 and the heparin binding human growth factors hVEGF and hKGF. The results summarized in Table 5 show that ligands 40-03 and 40-60 are specific for HTGFB I. Ligands 40-03 and 40-60 have binding affinities similar to random RNA to the other proteins tested.

These ligands are four to five orders of magnitude more specific for TGFB1 than even closely related proteins such as TGFB2 and other heparin binding growth factors. Of particular interest is the ability of these TGFB1 ligands to discriminate between TGFB1 and TGFB2 since these two proteins share 72% identity and are interchangeable in most biological assays (Roberts and Sporn (1991),"The Transforming Growth Factor-B's"in Peptide Growth Factors and Their Receptors, M. B. Sporn and A. B. Roberts, eds. (New York: Springer-Verlag)). Recently the solution three-dimensional structure of TGFB1 has been described and compared to the X-ray structure of TGFB2 (Hinck et al. (1996) Biochemistry 35: 8517-8534). Based on this comparison there is only a slight structural difference between TGFB1 and TGFB2 with a maximum root mean square deviation of 1.9 A (Hinck et al. (1996) Biochemistry 35: 8517-8534). BIAcore technology was also utilized to compare the binding specificity of ligands 40-03 and 40-60 between TGFB1 and TGFB2.

The analysis chip, loaded with either biotinylated 40-03, biotinylated 40-60, or biotinylated random RNA was exposed to various concentrations of TGFB1 or TGFB2 at 20 IlL/min in HBSMC-HSA, and data was collected during the association phase (3 min) and the dissociation phase (3 min).

Figures 3A-3F show a typical nested series of sensorgrams with TGFB1 and TGFB2 binding to random RNA, ligand 40-03 and ligand 40-60. These BIAcore results show that when applied at high concentrations, TGFB1 binds random RNA (Fig. 3A), ligand 40-03 (Fig. 3B) and ligand 40-60 (Fig. 3C) equivalently in a nonspecific manner with fast on-rates and off-rates. This non-specific binding is low affinity and non-stoichiometric, since stoichiometric binding would result in about 500 RU's of TGFB1 bound to the RNA on the chip (see Example l (f)). This non-specific binding represents the binding of random RNA

to TGFB1 also observed by nitrocellulose filter binding (see Example 2 (a)). When applied at lower concentrations, (less than 50 nM) TGFB1 binds ligand 40-03 and 40-60 but not random RNA. The specificity of TGFB1 for ligands 40-03 and 40-60 is mainly due to slower off rates compared to random RNA. This represents a specific interaction which appears to be stoichiometric, since the binding curves at this concentration plateau at about 400 RU's and the dissociation rates are very slow. See, for example, the triangles in Figure 3B, in which the dissociation rate is almost flat.

TGFB2 behaves differently in the same experiment. TGFB2 shows no binding to random RNA (Fig. 3D) and some binding to ligand 40-03 (Fig. 3E) and ligand 40-60 (Fig.

3F). This difference in binding affinity to random RNA is consistent with the increased negative charge content of TGFB2 compared to TGF131. The results in Figures 3D-3F clearly show that TGFB2 binds ligands 40-03 and 40-60 better than random RNA.

However, the observed TGFB2 binding to ligand 40-03 and 40-60 is still different, and lower than the corresponding binding of TGFB1. It seems that TGFB2 binds ligand 40-03 and 40- 60 with a very slow on and off rate suggesting induced fit. These results suggest that ligands 40-03 and 40-60 show cross-reactivity and bind to both TGFB1 and TGFB2 but with different affinities and kinetics.

Example 5. Inhibition of TGFBI bioactivity TGFB1 is a multifunctional growth factor (Roberts and Sporn (1991),"The Transforming Growth Factor-B's"in Peptide Growth Factors and Their Receptors, M. B.

Sporn and A. B. Roberts, eds. (New York: Springer-Verlag)). One of its activities is inhibition of proliferation of epithelial cells. For example, TGFB1 causes mink lung epithelial cells (MLEC) to cease replication, and it is manifested by reduction in 3H- thymidine incorporation. The midpoint of this response of MLEC is about 0.3 pM.

RNA from round 11 and 14 of the 40N and 3 ON pools along with random RNA controls were tested for TGF131 inhibitory activity using mink lung epithelial cells and measuring 3H-thymidine incorporation in the presence of 2 pM hTGFß 1. A significant HTGFBI inhibitory activity was observed with these advanced pools and not with random RNA (Figures 4A and 4B). It appears that the 40N round 14 pool was neutralizing serum- derived TGF131 in addition to the supplied TGFB1 since the amount of DNA synthesis at

high RNA concentrations is greater than that observed without exogenously added TGFB1 (Fig. 4A).

Using the same MLEC assay several individual ligands were screened for TGFB1 inhibitory activity. The results are summarized in Table 4 (Ki column). Several ligands were found that are good inhibitors of hTGFß 1. Typical results are shown in Figures 5A- 5D. It seems that the majority of good inhibitors belong in class 1 which contains only ligands from the 40N (Table 4, Ki column), and as expected, the best bioactivity correlated with binding activity.

TGFB1 proteins of various species are highly conserved proteins. The human and mouse or rat TGFB1 differ by a single amino acid. To determine the cross-species specificity, the ability of the TGFB1 ligands to inhibit the murine (m) TGFB I bioactivity was tested. Since mTGFBl is not commercially available, conditioned media from mouse cells was used. Several cell lines were screened for TGFB1 activity and it was found that 3T3 cells were the best source. Figure 6 shows the specificity of conditioned media used and the ability of ligand 40-03 and 40-60 to inhibit the bioactivity of such conditioned media.

Inhibition profiles with a pan-specific antibody (monoclonal mouse anti-TGFß 1/ TGFB2/TGFB3 antibody ; Fig. 6, open triangles) and a TGFB2/TGFB3 specific antibody (Fig. 6, open circles) demonstrate that the ability of the 3T3 conditioned media to inhibit the growth of MLEC is mainly due to TGFB1. Figure 6 also clearly demonstrates that, as expected, ligands 40-03 and 40-60 can inhibit the bioactivity of the 3T3 CM, presumably due to mTGFBl.

Example 6. Effect of library random region length on the outcome of the SELEX The above results suggest that size of the random region is important for the outcome of the SELEX process with TGFB1 in terms of obtaining bioactive ligands. These data are summarized in Table 6. It appears that the 30N pool contained ligands that bind TGFB1 with good affinities but these 30N ligands in general fail to inhibit the TGFB1 bioactivity.

The 20N pool failed to yield any TGFB1 ligands. Only the 40N pool yielded ligands that bind TGFB1 and inhibit its bioactivity.

Example 7. Proposed secondary structure ofbioactive TGFR1 Ligands The predicted common secondary structures among those ligands that could inhibit TGFBL bioactivity were investigated. These ligands appear to accommodate the proposed structure shown in Figure 7 which is a double pseudoknot. This structure is consistent with enzymatic digestion results obtained with three bioactive class 1 ligands. Such enzymatic digestion confirmed stem 1 and stem 2 while stem 3 was postulated on the basis of truncation results.

TABLE 1 Starting ssDNA templates 40N7: 5'GGGAGGACGATGCGG [-40N-] CAGACGACTCGCCCGA 3'SEQ ID NO: 1 30N7: 5'GGGAGGACGATGCGG [-30N-] CAGACGACTCGCCCGA 3'SEQ ID NO: 2 20N7: 5'GGGAGGACGATGCGG [-20N-] CAGACGACTCGCCCGA 3'SEQ ID NO: 3 SELEXPCRPrimers: 5G7: 5'TAATACGACTCACTATAGGGAGGACGATGCGG 3'SEQ ID NO: 4 3G7: 5'TCGGGCGAGTCGTCTG 3'SEQ ID NO: 5 TABLE 2. TGFB1 SELEX conditions and results Round [R]2,nM%B3S/N4PF5PB6Spin7Bf.U.nM Wash8 Wash9 40N <BR> <BR> 1A 100 5000 0. 42 13---5<BR> <BR> <BR> 1B 100 1000 0. 60 30. 7---5<BR> <BR> <BR> 2 100 500 0. 98 4. 9 +--5 3 100 500 3. 40 2. 6 +--10 4 100 500 4. 90 2. 9 +--10 5 33 167 2. 50 1. 9 +-+ 10 5 6 33 167 ND ND +-+ 10 55 <BR> <BR> 7 11 56 1. 00 8. 0 + + + 10 55 8 11 56 0. 40 5. 0 + + + 10 55 9 3. 3 16. 5 ND 13. 7 + + + 10 55 10 1. 1 5. 6 1. 55 16. 5 + + + 5 5 11 0. 33 1. 5 2. 00 7. 0 + + + 5 5 12* 0. 03 0. 15 1. 31 8. 0 + + + 5 5 13* 0. 0033 0. 016 0. 33 2. 4 + + + 5 5 14* 0. 011 0. 055 1. 00 3. 5 + + + 5 5 15L 0. 033 0. 0066 10. 00 130. 0 + + + 5 5 16L 0. 033 0. 0066 11. 50 345 + + + 5 5 30N <BR> <BR> 1A 140 7000 0. 36 4. 4---5<BR> <BR> <BR> 1B 140 1400 1. 80 20. 9---5<BR> <BR> <BR> 2 140 700 1. 90 11. 1 +--5<BR> <BR> <BR> 3 140 700 4. 60 4. 4 +--10<BR> <BR> <BR> 4 140 700 5. 20 9. 0 +--10 5 5. 0 25. 6 1. 50 4. 3 +-+ 10 5 6 11 55 0. 70 2. 6 +-+ 10 55 7 3. 3 16. 5 0. 26 1. 7 + + + 10 55 8 3. 3 16. 5 0. 10 2. 0 + + + 10 55 9 3. 3 16. 5 ND 14. 4 + + + 10 55 10 1. 1 5. 6 0. 39 4. 5 + + + 5 5 11 0. 33 1. 5 0. 38 4. 0 + + + 5 5 12* 0.03.15 0. 40 3. 0 + + + 5 5 13* 0.03.16 0. 49 3. 0 + + + 5 5 14 0.11.55 0. 90 10. 0 + + + 5 5 15 0. 033 0. 165 0. 50 6. 7 + + + 5 5 16L 0.11.022 1. 8 25. 7 + + + 5 5 17L 0. 033 0. 0066 1. 5 13. 6 + + + 5 5 Table 2 continued: Round [R]2,nM nM PF5PB6Spin7Bf.U.S/N4 Wash8Wash9 20N 1A 1000 50000 0.54 15.8---5 1B 100 1000 1.70 39.5---5 1C 1000 5000 3.80 51.0---5 2 1000 5000 3.70 72.5 +--5 3 1000 5000 5.90 122.0 +--10 4 330 1670 1.70 17.4 +--10 5 4.0 20.6 1.00 10.6 +-+ 10 5 6 1.2 6.1 0.60 4.7 +-+ 10 10 7 3.3 16.5 0.06 3.0 + + + 10 55 8 3.3 16.5 0. 30 15 + + + 10 55 9 3.3 16.5 ND 6.6 + + + 10 55 10 3.3 16.5 0. 31 16.5 + + + 5 5 11 1.1 5.6 0.19 4.0 + + + 5 5 12 1.1 5.6 1.2 13.0 + + + 5 5 13L 0.1 0.022 0.9 10.0 + + + 5 5 'Protein concentration in nanomolar 2RNA concentration in nanomolar 3Backround expressed as % of input 4Signal to noise 5Use of nitrocellulose prefiltered RNA 6Use of preblocked nitrocellulose with BSA 7Spinning of binding reactions before filtering through nitrocellulose 'Volume in ml of buffer wash 9Volume in ml of 0. 5M urea wash °L indicates RNA limiting SELEX conditions "The RNA pool used was a mixture of 2-3 pools obtained from 3 fold serial dilutions of a binding reaction. Only the most stringent condition is shown.

TABLE 3. Sequence of individual TGF#1 RNA ligands. The sequences of the fixed regions (Table 1) are not shown<BR> SEQ ID NO:<BR> 20-01 GUCUAUUUUUGCCUCCUCCC 6<BR> 20-02 AAUCCUUUCUUAAACCUCCC 7<BR> 20-03 UGUCUUUAGCUUAGGUUAUUCCUUCUGCCG 8<BR> 20-04 UGUCUUUAGCUUAGGUGAUUCCUUCUGCCG 9<BR> 20-05 UGUCUCUACCUUAGGUUGAUUCCUUCUACCG 10<BR> 20-06 UGAGUCUUGUUUUUUCGUC 11<BR> 20-07 UUGGCAUUGAAAGAGCUGGCAUACAUUCGC 12<BR> 20-08 UCCUUUCUAACAUUCCUCCC 13<BR> 20-09 GUCGUUGUUUUUCUCCUCCC 14<BR> 20-10 UGAGUCUUUCUUUUCGUCCC 15<BR> 20-11 GUCGUUUUUUGGUCCUC 16<BR> 20-12 GUUUUUAUUAUUCGUUUGGC 17<BR> 20-14 GUCGAUCAUUUUUAGCCUCCC 18<BR> 20-17 UGAGUUGAUCUUUUCGUCCC 19<BR> 20-18 UGCCUUUAGCUUAGGCAUUGCCUUCUGUG 20<BR> 20-19 CAAAAUUUUUGGUCAAGCCGUCAUUGCCGC 21<BR> 20-21 GUCGUUCUUUUUUCCCUCCC 22<BR> 20-23 AAUUUUUGUGAAGACGUUUGCCGCUUGCC 23<BR> 20-24 CGCAUCUUCUGUUUUCUCCC 24<BR> 20-25 GGAAUUUUUGGUAAAGCCGUAUGCCUCGC 25<BR> 20-26 UCAUCUCUGGGAGUUAAGAUCAUUUGGCCG 26<BR> 20-27 GCAGCCUCUGAUUUUCUCCC 27<BR> 20-28 GUCGUGAUUUUCGUUCUGCC 28<BR> 20-29 GUCGUAUUUUUUCCGCCUCCC 29<BR> 20-31 UCCUCAGCCUCUCACUUAUUAUCCUCCC 30<BR> 20-34 GUCUACUUGUUUUACCUCCC 31<BR> 20-35 CGAUUUUUUCGUCUUUUGGC 32<BR> 20-36 UGUCUAUAGCCUUGAUUAUAUCAUCUGCCG 33<BR> 20-37 CGAUUCCUCUUUUCACUCCC 34<BR> 20-38 UCCAUUUUUCUCCUCUCCC 35<BR> 20-40 GUUAAUUUUUGUCCUCUGGC 36<BR> 20-41 UUUUUUUCUUUUUUCUUUUUUUCCG 37 Table 3 cont'd<BR> SEQ ID NO:<BR> 20-42 UCGUCUUUGUUUUUCUCCC 38<BR> 20-43 UGUCUAUAGCCUUGAUUACAUCAUCUGCCG 39<BR> 20-45 UGCCUUUAGCUUAGGCAUUGCCUUCUGCCG 40<BR> 20-46 UGUCUAUAGCUUGAUUUUUAAUUUCUGCCG 41<BR> 20-47 UUUUAUUUUCUUCGUCUGGC 42<BR> 20-48 GAUGAACCGAACCGAGGUUAAGGUGCCAGAGUAGACGCUACU 43<BR> 20-49 UCGUCUAUUUUUUCCCUCCC 44<BR> 20-50 CUUUCGUCUGUUUUCCUGCC 45<BR> 30-01,07,18,23 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCG 46<BR> 30-02 CCUUGUUUUCUUUUUUCUUUUUUCACCCC 47<BR> 30-03 UGUCUUUAGCCCAGGUGAUUCCUUCUGCCG 48<BR> 30-04 UUAACCGUAAAGACGGCAUGAUGUAGUCCG 49<BR> 30-05 UUUUUUUAGCUUAGGUGAUUCCUUCNNCCU 50<BR> 30-06 UGCCUUUAGCUUAGGCUUUGCCUUCUGCCG 51<BR> 30-08 CGGAAUUUUUGUUGAGCCGUAUGCCGC 52<BR> 30-09,42 UGCCUUUAGCUUAGGUGAUUCCUUCUGCCG 53<BR> 30-10 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCG 54<BR> 30-12,24,21,40,41UGUCUAUAGCCUGAUUUUUAAUCUCUGCCG 55<BR> 30-15 UUGACCGUUAAGACGGCAUGAUGUGGUCCG 56<BR> 30-16,27,38,46 UGCCUUUAGCUUAGGCAUUGCCUUCUGCCG 57<BR> 30-17 UGCCUUUAGCUUAGGCUUUGCCUUCUGCCG 58<BR> 30-19 UUAACCNUAAAUACGGCUUGANUUCUUCCG 59<BR> 30-20 UGCCUUUAGCUUAGGCAUUGCCUUCUGCCG 60<BR> 30-22 UUAACCGUAAAGAGGGCAUGAUGUUUUCCG 61<BR> 30-25 UUGGCAUUGAAAGAGGCGUCAUAUGUUCGC 62<BR> 30-26 CCUUUCUUUCUUUUUAUUUUCUUCCCCUCCC 63<BR> 30-28 UGCCUUUAGCCUAGACCUUGUCUUCUGCCG 64<BR> 30-29 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCG 65<BR> 30-30 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCG 66<BR> 30-31 ACCGGUAAGGGCACUGCAGGAACACAAUCCCCUAUGCGAC 67<BR> 30-32 GGAAUUUUUGGUAAAGCCGUAUGCCUCGC 68<BR> 30-33 UGGCAUUGAAAGAGAUCGCAUACCUUCGC 69<BR> 30-34 UGUCUAUAGCCUUGAUUACAUCAUCUGCCU 70<BR> 30-35 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCU 71 Table 3 cont'd<BR> SEQ ID NO:<BR> 30-14 UGCCUUUAGCUUAUGCAUUGCCUUCUGCCG 72<BR> 30-36 UGCCUUUAGCUUAGGCAUUCGCCUUCUGCCG 73<BR> 30-37 UGUCUUUGGCCUAGGUGAUUCCUUCUGCCG 74<BR> 30-39 UGUCUUUAGCUUAGGUGAUUCCUUCUGCCG 75<BR> 30-43 UGUCUUUAGCCUAGGUGAUUCCUUCUGCCG 76<BR> 30-44 UGCCUUUAGCUUAGGCAUUGCCUUGCCG 77<BR> 30-45 GGUCUUUUAUUUUUUGUUUUUCUCUGUGCCC 78<BR> 30-47 UUAACCGUAAAGACAGCAUGAUGUAGUCUG 79<BR> 30-48 UUUUUUUCUUUUCCUUCCUUUUCUUACCG 80<BR> 30-49 UUAACCGUAAAGACGGCAUGAUGUUGUCCG 81<BR> 30-50 GGAAUUUUUGGUAAAGCCGUAUGCCUCGC 82<BR> 40-02 GCCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 83<BR> 40-03 GGGUUAUUGGGCGUCAACAUCCCCGAUUCUUUUCACGUC 84<BR> 40-04 AUGCCUUUUGCCUUCAGGGUGUAAUUCCUUGAUCUGUCCG 85<BR> 40-05 AACAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 86<BR> 40-06 UUAGGGGCGUCAACACCGCUAUCAUAAUUUUCGCCUUCCC 87<BR> 40-08 CGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCC 88<BR> 40-11 UGCCUUUAGUCUGAAUCUUCUACCAUGAUUCUCUGCCG 89<BR> 40-12 GACCCUUGUCUGCGAUUCAACUCGUAGGUUUUCUCACGUG 90<BR> 40-13 AGCAAGGUUACGAGGUCGGACCCUGCUGCCAACAUCCUCCC 91<BR> 40-14 CAUUAUGGCGUCAACAUGCCGGUUUUCGAUUCUCAUUGUC 92<BR> 40-15 CUCUAACUUCUUUUUCGCCUGUGUGUUUUCUUUUUGCUG 93<BR> 40-16 UUAGGGGCGUCAACACCGCUAUUACAUCUUUCGCCUCCC 94<BR> 40-17 GGUCGUUUUGUUUUUGUUUUUUGUAGCCCGGUCAUCCC 95<BR> 40-19 UUAGCGCGAGUUCAACACCGCAUGUGAUUCUUUCGCCUCC 96<BR> 40-20 UACAAGGUUACGCCGUCCGACCCUGCUGCCAACAUCCUCCC 97<BR> 40-21,34 GACCCUUGUCUGCGAUUCAACUCGUAGGUCUUCUCACGUG 98<BR> 40-22,35 UUAGGGGCGUCAACACCGCUAUUACAAUUUUCGCUUCC 99<BR> 40-23 UUAGGGGCGUCAACACCGCUAUUACAAUCUUCGCUUCC 100<BR> 40-24 UUAUGGGCGUCAACACCGCUAUUACAACUUUCGCUUUCC 101<BR> 40-25 UGUCGAUCGUUUGCUGUUUGAUUUCUUUUGUCCCUCCCGUG 102<BR> 40-26 UUAGGGGCGUCAACAUCGCUAUUACAAUCUUCGCCUUCC 103<BR> 40-28 UUAGGGGCGUCAACACCGCUAUUACAACUUUCGCCUCAC 104<BR> 40-29 GACCCUUUUCUGCGAUUCAACUCGUACGUCUUCUCACGUG 105 Table 3 cont'd<BR> SEQ ID NO:<BR> 40-31 UUAAGGGCGUCAACACCGCUAUUACAACUUUCGCUUCC 106<BR> 40-32 UUAUGGGCGUCAACACCGCUAUUACAACUUUCGCCUC 107<BR> 40-33 AGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 108<BR> 40-36 GUCAAGGUUACGCCGUCGGACCCUACUGCCCC 109<BR> 40-37 CUCCUAUAUUCAUGUUAUUGUUUUUUUCUUCCAGCUUGCCC 110<BR> 40-38 AGAUAAUUAUCAGCGGUGGACGGGGUGCCGGUACUGCCGC 111<BR> 40-39 UGCCUUUAGCCUAAGUUGAUCUAUUCAGCUUUCUGCCG 112<BR> 40-40 CCCAAGGUUACGCCGUCGGACCCUACUGCCAACUUCCUCCC 113<BR> 40-41 UGCCUUUAGCCUGAGUAUACUGAUGUAUAUUCUCUGCCG 114<BR> 40-42 UAGCGCGAGUUCAACACCGCAUGUGACUCUUUCGCCUCC 115<BR> 40-43 AUCCUUUUUUUAGCUUUUUUCUUUUUCCUGCCCCACUUCCC 116<BR> 40-44 UGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 117<BR> 40-45 GGGCUUUUCCUUUAGUACUUUUUUGUUUCGCUCCCCCC 118<BR> 40-51 UGCCUUUAGUCUGAAUCUUACCAUGCGAUUUUCUGCCG 119<BR> 40-52 AACAAGGUUACUCCGUCGGACCCUGCUGCCAACAUCCUCCC 120<BR> 40-53 GACUCUUGUCUGCGAUUCAACUCGUAGGUCUUCUCACGUG 121<BR> 40-54 UUAGGGGCGUCAACACCGCUAUCAUAACUUUCGCUUCCC 122<BR> 40-55 UUAGGGGCGUCAACACCGCUAUUCAACCUUCGCUUCCC 123<BR> 40-56 UUAGGGCGUCAACACCGCUAUUACAACUUUCGCCUCCC 124<BR> 40-57 GGUGUCGUCUUUCAACCCCU 125<BR> 40-58 UUAUGGGCGUCAACACCGCUAUUACAACUUUCGCCUCCC 126<BR> 40-59 CCCAAGGUUACGCCGUCGGACCCUGCUGCAAACAUCCUCCC 127<BR> 40-60 UUAUGGGCGUCAACACCGCUAUUACAGUUUUCGCCUCCCC 128<BR> 40-61,76 UUAGGGGCGUCAACACCGCUAUUACAAUCUUCGCUUUCC 129<BR> 40-62 GCCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCUUCCC 130<BR> 40-64 UUAGGGGCGUCAACACCGCUAUUACAAUCUUCGUCUUCC 131<BR> 40-65 GUCAAGUUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 132<BR> 40-66 UUCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 133<BR> 40-67 CUCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 134<BR> 40-68 UUAGGGGCUUCAACACCGCUAUUACAUUCUUCGCCUCCC 135<BR> 40-69 CACAAAGUUACGCCGUAGGACCCUGCUGCCAACAUCCUCCC 136<BR> 40-70 GGAUGGUCAGUUUCGGUUUUUCAUAUGUUUAUUUUCCCCCC 137<BR> 40-71 UAUUGACUUUUGUUUCUUUUUCUUUGCCUGGUCCC 138 Table 3 cont'd<BR> SEQ ID NO:<BR> 40-72 UUAGGGGCGUCAACACCGCUAUUACAACUUUCGCUUCCC 139<BR> 40-73 CUUCUUUUUCUUCUUUUCUUUAUGUCUUCUUCAUGCCG 140<BR> 40-75 GACCNUUGUNUGCGAUUCAACUCGUAGGUCUUCUCACGUG 141<BR> 40-77 UUAUGGGCGUCAACACCGCUAUUACAACUUUCGCCCCC 142<BR> 40-79 UUAUGGGUGUCAACACCGCUAUUACAACUUUCGCCUCCC 143 TABLE 4. Proposed alignment and observed affinity and bioactivity of TGFß1 ligands. The sequences of the fixed @<BR> Class 1<BR> Kd1 (nM) Kd2 (pM) P1(°@<BR> 40-03 GGGUUA UUGGGCGUCAACAUCCCCGAU UCUUUUCA CGUC 4.6~1.1 0.3~0.08 60.<BR> <P>40-06 UUA GGGGCGUCAACACCGCU AU CAUAAUUUU CGCCUUCCC 3.7~0.6 1.6~0.6 60.<BR> <P>40-14 CAUUA UGGCGUCAACAU GCCGGUUUUCGAUUCUCAUUGUC 5.7~1.4 0.4~0.2 62.<BR> <P>40-16 UUA GGGGCGUCAACACCGCU AU UACA UCUUU CGCCUCCC 1.7~0.6 0.06~0.04 48.<BR> <P>40-19 UUAGCGCGAGUUCAACACCGC AU GUGAUUCUUU CGCCUCC 4.2~2.2 3.7~2.7 68.<BR> <P>40-22, UUA GGGGCGUCAACACCGCU AU UACAAUUUU CGCUUCC 13.9~4.3 17.6~4.5 93.<BR> <P>35<BR> 40-23 UUA GGGGCGUCAACACCGCU AU UACAAUCUU CGCUUCC 14.2~4.5 15.6~4.0 58.<BR> <P>40-24 UUA UGGGCGUCAACACCGCU AU UACAACUUU CGCUUUCC 12.7~4.7 27.5~10.2 100<BR> 40-26 UUA GGGGCGUCAACAUCGCU AU UACAAUCUU CGCCUUCC 7.7~1.6 40.8~23.3 73.<BR> <P>40-28 UUA GGGGCGUCAACACCGCU AU UACAACUUU CGCCUCAC 12.3~2.4 81.6~47.2 93.<BR> <P>40-31 UUA AGGGCGUCAACACCGCU AU UACAACUUU CGCUUCC 8.4~2.8 0.7~0.4 34.<BR> <P>40-32 UUA UGGGCGUCAACACCGCU AU UACAACUUU CGCCUC 14.0~6.4 5.0~3.3 51.

40-42 UAGCGCGAGUUCAACACCGC AU GUGACUCUUU CGCCUCC 11.4~1.9 0.09 37.<BR> <P>40-54 UUA GGGGCGUCAACACCGCU AU CAUAACUUU CGCUUCCC 8.5~1.8 6.75~9 46.

40-55 UUA GGGGCGUCAACACCGCU AU U CAACCUU CGCUUCCC 8.0~2.5 0.1~0.06 64.<BR> <P>40-56 UUA GGGCGUCAACACCGCU AU UACAACUUU CGCCUCCC 4.2~1.3 0.2 41.<BR> <P>40-58 UUA UGGGCGUCAACACCGCU AU UACAACUUU CGCCUCCC 4.4~1.6 5.3~2.2 35.<BR> <P>40-60 UUA UGGGCGUCAACACCGCU AU UACAGUUUU CGCCUCCCC 3.8~1.5 1.9~0.8 32.<BR> <P>40-61, UUA GGGGCGUCAACACCGCU AU UACAAUCUU CGCUUUCC 13.5~4.2 1.0~0.8 56.<BR> <P>76<BR> 40-64 UUA GGGGCGUCAACACCGCU AU UACAAUCUU CGUCUUCC 5.6~2.0 3.1~1.8 37.<BR> <P>40-68 UUA GGGGCUUCAACACCGCU AU UACAUUCUU CGCCUCCC 20.7~3.2 0.4~0.3 62.<BR> <P>40-72 UUA GGGGCGUCAACACCGCU AU UACAACUUU CGCUUCCC 1.4~0.8 0.07~0.05 23.<BR> <P>40-77 UUA UGGGCGUCAACACCGCU AU UACAACUUU CGCCCCC 3.7~1.5 1.73~1.43 27.<BR> <P>40-79 UUA UGGGUGUCAACACCGCU AU UACAACUUU CGCCUCCC 0.6~0.1 0.5~0.4 18.

Table 4 cont'd<BR> Class 2<BR> Kd1 (nM) Kd2 (pM) P1(%) P<BR> 40-02 GCCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 14.8~1.4 100<BR> 40-05 AACAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 12.6~ 100<BR> 40-08 CGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCC 15.4~1.6 82<BR> 40-13 AGCAAGGUUACGAGGUCGGACCCUGCUGCCAACAUCCUCCC 15.4~9.6 100<BR> 40-20 UACAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 11.2~7.8 100<BR> 40-33 AGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 26.6~14 100<BR> 40-36 GUCAAGGUUACGCCGUCGGACCCUACUGCCCC 41.7~9.5 99<BR> 40-40 CCCAAGGUUACGCCGUCGGACCCUACUGCCAACUUCCUCCC 18~2.8 91.3<BR> 40-44 UGCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC 11.1~0.8 60.44<BR> 40-52 AACAAGGUUACUCCGUCGGACCCUGCUGCCAACAUCCUCCC<BR> 40-59 CCCAAGGUUACGCCGUCGGACCCUGCUGCAAACAUCCUCCC<BR> 40-62 GCCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCUUCCC<BR> 40-65 GUCAAGUUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC<BR> Kd1 (nM) Kd2 (pM) P1(%) P<BR> 40-66 UUCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC<BR> 40-67 CUCAAGGUUACGCCGUCGGACCCUGCUGCCAACAUCCUCCC<BR> 40-69 CACAAAGUUACGCCGUAGGACCCUGCUGCCAACAUCCUCCC<BR> Class 3<BR> Kd1 (nM) Kd2 (pM) P1(%) P<BR> 40-12 GACCCUUGUCUGCGAUUCAACUCGUAGGUUUUCUCACGUG 5.5~0.7 0.7~0.2 64.7 1<BR> 40-21, GACCUUGUCUGCGAUUCAACUCGUAGGUCUUCUCACGUG 10.1~3.5 6.5~4.0 100 9<BR> 34<BR> 40-29 GACCCUUUUCUGCGAUUCAACUCGUACGUCUUCUCACGUG 10.9~5.5 100<BR> 40-53 GACUCUUGUCUGCGAUUCAACUCGUAGGUCUUCUCACGUG<BR> 40-75 GACCNUUGUNUGCGAUUCAACUCGUAGGUCUUCUCACGUG Table 4 cont'd<BR> Class 4<BR> Kd1 (nM) Kd2 (pM) P1<BR> (%)<BR> 20-03 UGUCUUUAGCUUAGG UUAUUCCU UCUGCCG 0.11~0.1 0.3~1.7 9.3<BR> 20-04 UGUCUUUAGCUUAGG UGAUUCCU UCUGCCG 0.2 8.0<BR> 20-05 UGUCUCUACCUUAGG UUGAUUCCU UCUACCG 0.11~0.1 10.49<BR> 20-18 UGCCUUUAGCUUAGG CAUUGCC UUCUGUG<BR> 20-36 UGUCUAUAGCCUUGA UUAUAUCA UCUGCCG<BR> 20-43 UGUCUAUAGCCUUGA UUACAUCA UCUGCCG<BR> 20-45 UGCCUUUAGCUUAGG CAUUGCCU UCUGCCG<BR> 20-46 UGUCUAUAGCUUGAU UUUUAAUU UCUGCCG<BR> 30-01,07, UGUCUUUAGCCUAGG UGAUUCCU UCUGCCG 3.4 63<BR> 18,23<BR> 30-03 UGUCUUUAGCCCAGG UGAUUCCU UCUGCCG 58.2 262 #100<BR> 30-05 UUUUUUUAGCUUAGG UGAUUCCU UCNNCCU 100<BR> 30-06 UGCCUUUAGCUUAGG CUUUGCCU UCUGCCG 56.5 74.1 #100<BR> 30-09,42 UGCCUUUAGCUUAGG UGAUUCCU UCUGCCG 5.35~0.9 22.7<BR> 30-10 UGUCUUUAGCCUAGG UGAUUCCU UCUGCCG 4.05~1.5 75.5 28.6<BR> 30-12,24, UGUCUAUAGCCUGAU UUUUAAUC UCUGCCG 2.82~1.7 60.8 24.8<BR> 21,40,41<BR> 30-14 UGCCUUUAGCUUAUG CAUUGCCU UCUGCCG<BR> 30-16,27, UGCCUUUAGCUUAGG CAUUGCCU UCUGCCG 71.8 211~90 99.8<BR> 38,46<BR> 30-17 UGCCUUUAGCUUAGG CUUUGCCU UCUGCCG 67 205~93 99.9<BR> 30-20 UGCCUUUAGCUUAGG CAUUGCCU UCUGCCG 2.57~0.3 0.7 22.9<BR> 30-28 UGCCUUUAGCCUAGA CCUUGUCU UCUGCCG 0.78~.07 0.4~0.2 17.9<BR> 30-29 UGUCUUUAGCCUAGG UGAUUCCU UCUGCCG<BR> 30-30 UGUCUUUAGCCUAGG UGAUUCCU UCUGCCG<BR> 30-34 UGUCUAUAGCCUUGA UUACAUCA UCUGCCU 9.5~1.6 68.5<BR> 30-35 UGUCUUUAGCCUAGG UGAUUCCU UCUGCCU 20.7~13.8 224~123 90.3<BR> 30-36 UGCCUUUAGCUUAGG CAUUCGCCUUCUGCCG 3.9~1.1 46.6<BR> 30-37 UGUCUUUGGCCUAGG UGAUUCCU UCUGCCG 2.65~0.7 46.6<BR> 30-39 UGUCUUUAGCUUAGG UGAUUCCU UCUGCCG 6.02~1.5 32.5~16.2 48.5<BR> 30-43 UGUCUUUAGCCUAGG UGAUUCCU UCUGCCG 50.5 100 Table 4 cont'd<BR> Kd1 (nM) Kd2 (pM) P1<BR> (%)<BR> 30-44 UGCCUUUAGCUUAGG CAUUGC CUUGCCG<BR> 40-04 AUGCCUUUUGCCUUCAGGGUGU AAUUCCUUGAUC UGUCCG 5.09~0.6 1.4~1.2 56.7<BR> 40-11 UGCCUUUAGUC UGAAUCUUCUACCA UGAUUC UCUGCCG 4.6~0.7 4.9~3.1 68.9<BR> 40-39 UGCCUUUAGCC UAAGUUG AUCUAUUCAGCUU UCUGCCG 11.5~2.0 8.78~6.4 64.2<BR> 40-41 UGCCUUUAGCC UGAGUAU ACUGAUGUAUAUUC UCUGCCG 3.8~0.9 1.4~1.1 30.8<BR> 40-51 UGCCUUUAGUC UGAAUCUU ACCAUGCGAUUU UCUGCCG<BR> Class 5<BR> Kd1 (nM) Kd2 (pM) P1@<BR> 20-26 UCAUCUCUGGGAGUUAAGAUCAUUUGGCCG<BR> 30-04 UUAACCGUAAAGACGGCAUGAUGUAGUCCG 5.03~0.8 33.<BR> <P>30-15 UUGACCGUUAAGACGGCAUGAUGUGGUCCG 55.3 95.<BR> <P>30-19 UUAACCNUAAAUACGGCUUGANUUCUUCCG 5.7~1.9 346 28.

30-22 UUAACCGUAAAGACGGCAUGAUGUUUUCCG 2.47 32.<BR> <P>30-47 UUAACCGUAAAGACAGCAUGAUGUAGUCUG<BR> 30-49 UUAACCGUAAAGACGGCAUGAUGUUGUCCG Class 6<BR> Kd1 (nM) Kd2 (pM) P1(@<BR> 20-19 CAAAAUUUUUGGUCAAGCCGUCAUUGCCGC<BR> 20-23 AA UUUUUGUGAAGACGUU UGCCGCUUUGCC<BR> 20-25 GGAAUUUUUGGUAAAGCCG UA UGCCUCGC<BR> 30-08 CGGAAUUUUU GUUGAGCCG UA UGCCGC 10.2~2.9 33.<BR> <P>30-32 GGAAUUUUUGGUAAAGCCG UA UGCCUCGC<BR> 30-50 GGAAUUUUUGGUAAAGCCG UA UGCCUCGC<BR> Class 7<BR> Kd1 (nM) Kd2 (pM) P1(@<BR> 20-07 UUGGCAUUGAAAGAGCUGGCAUACAUUCGC<BR> 30-25 UUGGCAUUGAAAGAGGCGUCAUAUGUUCGC 23~6.4 1.14~.6 79<BR> 30-33 UGGCAUUGAAAGAGAUCGCAUACCUUCGC Table 4 cont'd<BR> Class 8<BR> Kd1 (nM) Kd2 (pM) P1(@<BR> 20-48 GAUGAACCGAACCGAGGUUAAGGUGCCAGAGUAGACGCUCAU<BR> 30-31 ACCGGUAAGGGCACUGCAGGAACACAAUCCCCUAUGCGAC 100<BR> 40-38 AGAUAAUUAUCAGCGGUGGACGGGGUGCCGGUACUGCCGC 21.8~5.3 92.<BR> <P>Class 9<BR> Kd1 (nM) Kd2 (pM) P1<BR> (%)<BR> 20-01 GUCUAUUUUU GCCUCCUCCC<BR> 20-02 AAUCCUUUCUUAAA CCUCCC 0.6 5<BR> 20-06 UGAGUCUUGUUUUUU CGUC 0.3 5<BR> 20-08 UCCUUUCUAACAUU CCUCCC 20-09 GUCGUUGUUUUU CUCCUCCC<BR> 20-10 UGAGUCUUUCUUUU CGUCCC<BR> 20-11 GUCGUUUUUUU GGUCCUC<BR> 20-12 GUUUUUAUUAUUCGUUUGGC 20-14 GUCGAUCAUUUUU AGCCUCCC<BR> 20-17 UGAGUUGAUCUUUU CGUCCC<BR> 20-21 GUCGUUCUUUUUU CCCUCCC<BR> 20-24 CGCAUCUUCUGUUUUCU CCC<BR> 20-27 GCAGCCUCUGAUUUUCU CCC<BR> 20-28 GUCGUGAUUUU CGUUCUGCC<BR> 20-29 GUCGUAUUUUUU CCGCCUCCC<BR> 20-31 UCCUCAGCCUCUCACUUAUUAUCCUCCC<BR> 20-34 GUCUACUUGUUUU ACCUCCC<BR> 20-35 CGAUUUUUUCGUCUUUUG GC<BR> 20-37 CGAUUCCUCUUUUC ACUCCC<BR> 20-38 UCCCAUUUUU CUCCUCUCCC<BR> 20-40 GUUAAUUUUUGUCCUCUGGC<BR> 20-41 UUUUUUUCUUUUUUCUUUUUUU CCG<BR> 20-42 UCGUCUUUGUUUUU CUCCC<BR> 20-47 UUUUAUUUUCUU CGUCUGGC Table 4 cont'd<BR> Kd1 (nM) Kd2 (pM) P1<BR> (%)<BR> 20-49 UCGUCUAUUUUUU CCCUCCC<BR> 20-50 CUUUCGUCUGUUUU CCUGCC<BR> 30-02 CCUUGUUUUCUUUUUUCUUUUUUCACCCC 5.3~2.8 0.8~0.9 27.1<BR> 30-26 CCUUUCUUUCUUUUUAUUUUCUU CCCCUCCC<BR> 30-45 GGUCUUUUAUUUUUUGUUUUUCU CUGUGCCC<BR> 30-48 UUUUUUUCUUUUCCUUCCUUUUCUUACCG<BR> 40-15 CUCUAACUUCUUUUUCGCCUGUGUGUUUUCUUUUU GCUG<BR> 40-17 GGUCGUUUUGUUUUUGUUUUUUGUAGCCCGGUCAUCCC<BR> 40-25 UGUCGAUCGUUUGCUGUUUGAUUUCUUUU GUCCCUCCCGUG<BR> 40-37 CUCCUAUAUUCAUGUUAUUGUUUUUUUCUU CCAGCUUGCCC<BR> 40-43 AUCCUUUUUUUAGCUUUUUUCUUUUU CCUGCCCCACUUCCC 2.2~1.7 12<BR> 40-45 GGGCUUUUCCUUUAGUACUUUUUUGUUU CGCUCCCCCC<BR> 40-57 GGUGUCGUCUUUC AACCCCU 40-70 GGAUGGUCAGUUUCGGUUUUU CAUAUGUUUAUUUUCCCCCC<BR> 40-71 UAUUGACUUUUGUUUCUUUUUCUUUGCCUGGUCCC<BR> 40-73 CUUCUUUUUCUUCUUUUCUUUAUGUCUUCUU CAUGCCG Table 4 continued<BR> Kd1 = Dissociation rate constant in nanomolar of the low affinity component of biphasic binding curves or dissociati@<BR> of monophasic binding curves<BR> Kd2 = Dissociation rate constant in picomolar of the high affinity component of biphasic binding curves<BR> P1 = Plateau values in % of monophasic curves or of the low affinity component of biphasic curves<BR> P2 = Plateau values in % of the high affinity component of biphasic curves<BR> P2/P1 = Fraction in % of the high affinity component of biphasic curves<BR> Ki = Inhibition constant in nanomolar obtained from the MLEC assay<BR> BCG = Nitrocellulose binding background expressed as % of input TABLE 5. Binding Specificity of TGFßl Ligands 40-03 and 40-60 Target KDhTGFß1KDTarget/KDhTGFß1/ 40-03 40-60 1hTGFß11 hTGFB2 >340,000 >340,000 hKGF >34,000 >34,000 hVEGF >340,000 >340,000 When applicable, the high affinity component of biphasic binding was used.

TABLE 6. Results of TGFß1 SELEX with random regions of 20 30 and 40N expressed by the distribution<BR> of ligands in the different classes and the binding and inhibitory activity of these classes<BR> SELEX Pools Affinities<BR> 40N 30N 20N Biph1. KD#pM2 Ki3<BR> Total clones 64 48 40<BR> Unique clones 61 37 40<BR> Class 1 39.3% + + +++<BR> Class 2 26.2% - - -<BR> Class 3 8.2% + + ++<BR> Class 4 8.2% 56.7% 20.0%4 + + ~<BR> Class 5 16.2% 2.5%4 + + +<BR> Class 6 8.1% 7.5%4 - - ND<BR> Class 7 5.4% 2.5%4 ~ - ND<BR> Class 8 1.6% 2.7% 2.5%4 - - ND<BR> Class 9 16.4% 10.8% 65.0% NC5 -<BR> Length of 40 59 (96.7%) 1 (2.7%) 1 (2.5%)<BR> Length of 30 1 (1.6%) 36 (97.3%) 15 (37.5%)<BR> Length of 20 1 (1.6%) 24 (60.0%)<BR> 1Biphasic binding is shown by plus (+), monophasic by minus (-), and unclear results by plus/minus (~)<BR> 2Low pmolar KD values are shown by plus (+) and KD values similar to random RNA are shown by minus (-)<BR> 3High, intermediate, low, and possible bioactivity is shown by 3 pluses (+++), two pluses (++),<BR> one plus (+) or plus/minus (~), respectively<BR> 4longer than 20N<BR> 5nitrocellulose binders