High-affinity oligonucleotide ligands to secretory phospholipase A2 (sPLA.sub.2)

FIELD OF THE INVENTION
Described herein are methods for identifying and preparing high-affinity nucleic acid ligands to secretory phospholipase A2 (sPLA.sub.2). The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. Specifically disclosed herein are high-affinity nucleic acid ligands. The invention includes high-affinity RNA ligands which bind to sPLA.sub.2 and inhibit the enzymatic activity of sPLA.sub.2.
BACKGROUND OF THE INVENTION
Inflammation is a local response to tissue damage or foreign material which is designed to isolate and/or destroy injured tissues and foreign substances. Uncontrolled inflammatory responses may irreparably damage host tissue, as in the chronic inflammation of rheumatoid arthritis or lead to multiple organ failure and death as in the systemic inflammation of septic shock.
Eicosanoids are metabolic products of the essential fatty acid, arachadonic acid and are known to play a central role in the inflammatory response. Arachadonic acid is an integral component of cell membranes where it is commonly found as an sn-2 acyl or akyl ester of 3-sn-phosphoglycerides. Phospholipase A2 (PLA.sub.2) is a class of enzymes that specifically catalyze the hydrolysis of the sn-2 acyl or alkyl ester of phosphoglycerides, producing equimolar quantities of lysophospholipids and free fatty acids. The PLA.sub.2 catalyzed hydrolysis of one such membrane phospholipid, alkyl-arachidonyl-glycerophosphatidylcholine, yields free arachidonic acid and lyso-platelet activating factor (lyso-PAF), the precursor of PAF, in equimolar amounts. Since the availability of free arachidonic acid is rate limiting for eicosanoid synthesis, the pro-inflammatory role of PLA.sub.2 is thought to be the consequence of its role in arachadonic acid metabolism.
Three mammalian PLA.sub.2 enzymes are known. Pancreatic PLA.sub.2 is related to the type I PLA.sub.2 from the venom of Elapidae and Hydrophide both in primary and tertiary structure. A digestive enzyme, synthesized as a proenzyme in the pancreas, it is unlikely to play a central role in inflammatory conditions.
Two non-pancreatic mammalian enzymes have been described. One is a high molecular weight intracellular enzyme. The other is a soluble enzyme, sPLA.sub.2, and is of particular interest because it has been isolated from inflammatory exudates, such as synovial fluid (Seilhamer et al. (1989) J. Biol. Chem. 264: 5335-5338) and from platelets (Kramer et al. (1989) J. Bio. Chem. 264: 5768-5775). sPLA.sub.2 is known by several equivalent names, including secretory phospholipase A.sub.2, soluble phospholipase A.sub.2 and synovial phospholipase A2, all of which can be used interchangeably. This enzyme, which has been sequenced, has a molecular weight of 14 kD, a pI>10 and is homologous to type II PLA.sub.2 from the venoms of Crotalidae and Viperidac (Kramer et al. (1989) supra; Scott et al. (1991) Science 254: 1007; Wery et al. (1991) Nature 352: 79-82).
The involvement of sPLA.sub.2 in the inflammatory response is supported by two types of data. First, elevated levels of serum PLA.sub.2 activity have been observed in diseases such as endotoxemia (Vadas and Hay (1983) Can. J. Physiol. Pharmacol. 61: 561-566), sepsis (Vadas (1984) J. Lab. Clin. Med. 104: 873-881; Nevalainen et al. (1992) Clin. Chem. 38: 1824-1829), rheumatoid arthritis (Pruzanski et al. (1988) J. Rheumatol. 15: 1351-1355), pancreatitis (Nevalainen et al. (1993) Gut 34: 1133-1136) and uremia (Costello et al. (1990) Clin. Chem. 36: 198-200) and in some studies correlate with severity and outcome. One of the best documented cases is acute pancreatitis in which sPLA.sub.2 immuno-reactive activity, but not pancreatic PLA.sub.2 immuno-reactive activity, is correlated with serum PLA.sub.2 enzymatic activity. The duration of the elevated levels is longer for the more severe necrotizing pancreatitis than the less severe oedmatous form (Nevalainen et al. (1993) supra).
Second, in both animal and tissue models (Snyder et al. (1993) J. Pharmacol. and Therapeutics 266: 1147-1155), the introduction of sPLA.sub.2 results in an inflammatory like response.
The isolation of specific antagonists to sPLA.sub.2 would have multiple uses. First, sPLA.sub.2 ligands would provide a useful tool for defining the enzyme's role in inflammatory responses and in diagnosing various inflammatory conditions. Second, sPLA.sub.2 antagonists would be useful as an anti-inflammatory therapeutic. Given the cationic nature of sPLA.sub.2 and the specificity of high affinity oligonucleotide ligands, SELEX technology is well suited for the isolation of sPLA.sub.2 antagonists which would not cross react with pancreatic or high molecular weight PLA2s. The present invention demonstrates the successful isolation of high-affinity oligonucleotide antagonists to sPLA.sub.2.
BRIEF SUMMARY OF THE INVENTION
The present invention includes methods of identifying and producing nucleic acid ligands to secretory phospholipase A2 (sPLA.sub.2) and the nucleic acid ligands so identified and produced. More particularly, RNA sequences are provided that are capable of binding specifically to sPLA.sub.2.
Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to sPLA.sub.2 comprising the steps of (a) preparing a candidate mixture of nucleic acids, (b) partitioning between members of said candidate mixture on the basis of affinity to sPLA.sub.2, and (c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to sPLA.sub.2.
More specifically, the present invention includes the RNA ligands to sPLA.sub.2 identified according to the above-described method, including those ligands listed in Table 2. Also included are RNA ligands to sPLA.sub.2 that are substantially homologous to any of the given ligands and that have substantially the same ability to bind sPLA.sub.2 and antagonize sPLA.sub.2 activity. Further included in this invention are RNA ligands to sPLA.sub.2 that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind sPLA.sub.2 and antagonize sPLA.sub.2 activity.
The present invention also includes modified nucleotide sequences based on the RNA ligands identified herein and mixtures of the same.
DETAILED DESCRIPTION OF THE INVENTION
This application describes high-affinity nucleic acid ligands to sPLA.sub.2 identified through the method known as SELEX. SELEX is described in U.S. patent application Ser. No. 07/536,428, entitled Systematic Evolution of Ligands by EXponential Enrichment, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled Nucleic Acid Ligands, now U.S. Pat. No. 5,270,163, (see also PCT/US91/04078). These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
In its most basic form, the SELEX process may be defined by the following series of steps:
1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (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 a 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 unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
The SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate an 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.
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 sPLA.sub.2. In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligands to sPLA.sub.2 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 co-pending and commonly assigned U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992 ('624), now U.S. Pat. No. 5,496,938, methods are described for obtaining improved nucleic acid ligands after SELEX has been performed. The '624 application, entitled Nucleic Acid Ligands to HIV-RT and HIV-1 Rev, is specifically incorporated herein by reference.
In the present invention, a SELEX experiment was performed in search of RNA with specific high affinity for sPLA.sub.2 from a degenerate library containing 30 or 50 random positions (30N or 50N). This invention includes the specific RNA ligands to sPLA.sub.2 shown in Table 2 (SEQ ID Nos.:10-122), identified by the methods described in Examples 1 and 2. The scope of the ligands covered by this invention extends to all nucleic acid ligands of sPLA.sub.2, modified and unmodified, identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to the ligands shown in Table 2. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. A review of the sequence homologies of the ligands of sPLA.sub.2 shown in Table 2 shows that sequences with little or no primary homology may have substantially the same ability to bind sPLA.sub.2. For these reasons, this invention also includes nucleic acid ligands that have substantially the same ability to bind sPLA.sub.2 as the nucleic acid ligands shown in Table 2. Substantially the same ability to bind sPLA.sub.2 means that the affinity is within a few orders of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence--substantially homologous to those specifically described herein--has substantially the same ability to bind sPLA.sub.2.
This invention also includes the ligands as described above, wherein certain chemical modifications are made in order to increase the in vivo stability of the ligand or to enhance or mediate the delivery of the ligand. Examples of such modifications include chemical substitutions at the sugar and/or phosphate and/or base positions of a given nucleic acid sequence. See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled High Affinity Nucleic Acid Ligands Containing Modified Nucleotides, now abandoned, specifically incorporated herein by reference. 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.
The nucleic acid ligands to the sPLA.sub.2 protein described herein are useful as pharmaceuticals and as diagnostic reagents.

EXAMPLE 1
Experimental Procedures
A. Materials
The human sPLA.sub.2, rabbit anti-human sPLA.sub.2 polyclonal antibody, and enzymatic and chromogenic substrates used in these experiments were supplied by Eli Lilly & Co. hen egg-white lysozyme (6.times. crystallized) was provided by Dr. S. C. Gill, Jr and is commercially available. The 2' NH.sub.2 modified CTP and UTP were prepared according to Pieken et al. (1991) Science 253: 314-317. DNA oligonucleotides were synthesized by Operon. All other reagents and chemicals were purchased from commercial sources.
B. SELEX: 2'OH RNA
The SELEX procedure is described in detail in U.S. Pat. No. 5,270,163 and elsewhere. The DNA template used in 2'OH RNA SELEXes contained 30 random nucleotides, flanked by the N1 template 5' and 3' fixed regions (30N1 (SEQ ID NO.:1), Table I). The fixed regions include DNA primer annealing sites for PCR and cDNA synthesis as well as the consensus T7 promoter region to allow in vitro transcription. These single-stranded DNA molecules were converted into double-stranded transcribable templates by PCR amplification using the primers indicated in Table I (SEQ ID NOs.:2 and 3). PCR conditions were 50 mM KCl, 10 mM Tris-Cl, pH 9, 0.1% Triton X-100, 7.5 mM MgCl.sub.2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and contained 25 U/ml of Taq DNA polymerase. Transcription reactions contained 5 mM DNA template, 5 U/.mu.l T7 RNA polymerase, 40 mM Tris-Cl (pH 8.0), 12 mM MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100, 4% PEG 8000, 2 mM each of 2'OH ATP, 2'OH GTP, 2'OH CTP, 2'OH UTP, and 0.31 mM a .sup.32 P 2'OH ATP. SELEX binding reactions are outlined in Table I. For the binding reactions, RNA molecules were incubated with sPLA.sub.2 in TBSC buffer (137 mM NaCl, 5 mM KCl, 5 mM CaCl.sub.2, 10 mM Tris-HCl, pH 7.4) for 15 minutes at 37.degree. C. Protein-RNA complexes were separated from unbound RNA by nitrocellulose filter partitioning and bound RNA was isolated from filters by phenol/urea extraction. The RNA was reverse transcribed into cDNA by AMV reverse transcriptase at 48.degree. C. for 60 minutes in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6 mM Mg(OAc).sub.2, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 U/.mu.l AMV RT. PCR amplification of this cDNA resulted in approximately 500 pmol double-stranded DNA, transcripts of which were used to initiate the next round of SELEX.
C. SELEX: 2'NH.sub.2 RNA
For 2'NH.sub.2 SELEXes, DNA templates contained 50 random nucleotides flanked by N7 (50N7 SELEX (SEQ ID NO.:4)) or by N9 (50N9 SELEX (SEQ ID NO.:7)) 3' and 5' fixed regions shown in Table I. The RNAs transcribed from these templates contained cytidine and uridine in which the 2'OH of the ribose moiety was replaced with a NH.sub.2 group. In these SELEXes, both nitrocellulose partitioning (50N9 SELEX: rounds 6, 7, 10 and 11; 50N7 SELEX rounds 7, 8, 11 and 12) and sPLA.sub.2 immobilized on beads via an anti-sPLA.sub.2 polyclonal antibody (50N9 SELEX: rounds 1-5, 8 and 9; 50N7 SELEX rounds 1-6, 9 and 10) were used to separate free from bound RNA.
Polyclonal anti-sPLA.sub.2 agarose beads were prepared from an ammonium sulfate precipitated, anion exchange purified immunoglobulin fraction of an sPLA.sub.2 immunized rabbit. The immunoglobulins were bound to hydrazide activated agarose beads (CARBOLINK Coupling Gel, Pierce, Rockford, Ill.) according to the manufacturer's instructions. The resulting immunoglobulin density was estimated to be 1.2 mg/ml of gel. Coupling of sPLA.sub.2 was accomplished by incubating 50 .mu.l of Ab-gel with 500 .mu.l of 2 .mu.M sPLA.sub.2 in TBSC for 2 hours at 37 .degree. C. The washed gel, which was resuspended in 500 .mu.l of TBSC and stored at 4.degree. C., had a calculated sPLA.sub.2 density of 0.2 to 2 pmol/.mu.l of gel, assuming that 1 to 10% of the Ig fraction is anti-sPLA.sub.2 Ab and a stoichiometry of 2 molecules of sPLA.sub.2 bound per antibody molecule.
For rounds in which immobilized sPLA.sub.2 was used to partition unbound RNA from sPLA.sub.2 /RNA complexes, RNA was incubated with washed sPLA.sub.2 -gel in a siliconized column for 5 minutes at 37.degree. C., as indicated in Table I. Unbound RNA was removed by extensive washing with TBSC. Bound RNA was eluted as two fractions; the first fraction was eluted with calcium free buffer, TBS; the second fraction was eluted with free polyclonal anti-sPLA.sub.2 Ab in TBS and was processed for use in the following round. The percentage of input RNA eluted by each step is recorded in Table I. For rounds in which partitioning was accomplished by nitrocellulose filter binding (Table I), free sPLA.sub.2 and RNA were incubated for 5 minutes at 37.degree. C., filtered through TBSC prewashed nitrocellulose filters and then washed with 3 ml of TBSC.
RNA/sPLA.sub.2 complexes absorbed to nitrocellulose filters and fractions eluted from immobilized sPLA.sub.2 were heated at 90.degree. C. for 5 minutes in 1% SDS, 2% b-mercaptoethanol and extracted with phenol/chloroform. The RNAs were then processed as in the 2'OH SELEXes except that 2'NH.sub.2 CTP and 2'NH.sub.2 UTP were substituted for CTP and UTP in transcription reactions.
D. Nitrocellulose Filter Partitioning
The nitrocellulose filter partitioning method was used as described in SELEX Patent Applications to determine the affinity of RNA ligands for sPLA.sub.2 and for other promins. Filter discs (nitrocellulose/cellulose acetate mixed matrix, 0.45 .mu.m pore size, Millipore) were placed on a vacuum manifold and washed with 5 ml of TBSC buffer under vacuum. Reaction mixtures, containing .sup.32 P labeled RNA pools and sPLA.sub.2, were incubated in TBSC for 5 minutes at 37.degree. C., filtered, and then immediately washed with 5 ml TBSC. The filters were air-dried and counted in a Beckman liquid scintillation counter without fluor.
The equilibrium dissociation constant, K.sub.d, for an RNA pool or specific ligand that binds monophasically is given by the equation
K.sub.d =[P.sub.f ][R.sub.f ]/[RP]
where,
[Rf]=free RNA concentration
[Pf]=free Protein concentration
[RP]=concentration of RNA/protein complexes
K.sub.d =dissociation constant
A rearrangement of this equation, in which the fraction of RNA bound at equilibrium is expressed as a function of the total concentration of the reactants, was used to calculate K.sub.d s of monophasic binding curves:
q=(P.sub.T +R.sub.T +K.sub.d -((P.sub.T +R.sub.T +K.sub.d).sup.2 -4 P.sub.T R.sub.T).sup.1/2)
q=fraction of RNA bound
[P.sub.T ]=total protein concentration
[R.sub.T ]=total RNA concentration
Many ligands and evolved RNA pools yield biphasic binding curves. Biphasic binding can be described as the binding of two affinity species that are not in equilibrium. Biphasic binding data were evaluated with the equation
q=2P.sub.t +R.sub.t +K.sub.d1 +K.sub.d2 -[(P.sub.t +X.sub.1 R.sub.1 +K.sub.d1).sup.2 -4P.sub.t X.sub.1 R.sub.t ].sup.1/2 -[(P.sub.t +X.sub.2 R.sub.t +K.sub.d2).sup.2 -4P.sub.t X.sub.2 R.sub.t ].sup.1/2,
where X.sub.1 and X.sub.2 are the mole fractions of affinity species R.sub.1 and R.sub.2 and K.sub.d1 and K.sub.d2 are the corresponding dissociation constants. K.sub.d s were determined by least square fitting of the data points using the graphics program Kaleidagraph (Synergy Software, Reading, Pa.).
E. Cloning and Sequencing
During the last round of SELEX, PCR of cDNA was performed with primers which contain recognition sites for the restriction endonucleases HindIll and BamHI. Using these restriction sites, the DNA sequences were inserted directionally into the pUC 18 vector. These recombinant plasmids were transformed into E. coli strain XL-1 Blue (Stratagene, La Jolla, Calif.). Plasmid DNA was prepared according to the alkaline hydrolysis method (Zhou et al. (1990) Biotechniques 8: 172-173) and about 100 clones were sequenced with the Sequenase sequencing kit (United States Biochemical Corporation, Cleveland, Ohio).
F. Ligand Truncation
Truncation experiments were carried out to determine the minimal sequence necessary for high affinity binding of the RNA ligands to sPLA.sub.2. For 3' boundary determination, RNA ligands were 5' end-labeled with Y .sup.32 P-ATP using T4 polynucleotide kinase. 5' boundaries were established with 3' end-labeled ligands using a .sup.32 P-pCp and T4 RNA ligase. After partial alkaline hydrolysis, radiolabeled RNA ligands were incubated with sPLA.sub.2 at concentrations ranging from 0.04 nM to 28 nM and sPLA.sub.2 /RNA complexes were separated from unbound RNA by nitrocellulose partitioning. RNA truncates that bound with high affinity were identified on high-resolution denaturing polyacrylamide gels. For each radioactively labeled ligand, two types of ladders were generated to serve as markers: 1) a partial alkaline hydrolysis ladder, and 2) a partial RNase T1 digestion ladder.
EXAMPLE 2
RNA Ligands to sPLA.sub.2
A. 2'OH SELEX
The primary objectives of the 2'OH SELEXes were 1) to generate high affinity ligands to human sPLA.sub.2 and 2) to determine if a non-amplifiable, non-specific competitor could provide selection pressure for high affinity ligands under experimental conditions which, in the absence of competitor, did not detectably enrich for high affinity ligands. Experimental conditions for the Control, Competition, and Standard SELEX procedures are outlined in Table 1. The (Control) SELEX experiment was initiated with 240 nM sPLA.sub.2 and a 10-fold molar excess of 2'OH RNA randomized at 30 contiguous positions, 30N1 (SEQ ID NO.:1); these conditions were maintained throughout this SELEX. A second (Competition) SELEX, in which increased stringency was imposed by a high concentration of a non-amplifiable competitor (0.5 mM tRNA), was initiated with an aliquot of the third round Control SELEX RNA. A third (Standard) SELEX, in which stringency was increased by reducing the sPLA.sub.2 concentration to 12 nM, while maintaining a 10-fold molar excess of RNA, was started with an aliquot of the fifth round Control SELEX RNA.
The starting pool contained approximately 5.times.10.sup.14 RNA molecules (500 pmol) and bound sPLA.sub.2 with K.sub.d of 240 nM. After 12 rounds, the Standard and Competition SELEX pools bound sPLA.sub.2 biphasically; the high affinity species represented about 25% of the molecules in both pools and bound with a K.sub.d of 4 nM. No improvement was observed in the affinity of the Control SELEX RNA. These results validate the non-amplifiable competitor strategy.
The three SELEXes showed no further improvement in affinity in subsequent rounds. Twelfth and fourteenth round cDNAs from the Standard and Competition SELEXes, respectively, were PCR amplified and cloned into pUC 18; 51 clones from the Standard and 34 from the Competition SELEX were sequenced. Sequences were aligned manually and are provided in Table II. The sequences from the 2'OH SELEX from 30N1 RNA are designated by ligand names with numbers only (SEQ ID NOs.:10-59).
B. 2'NH.sub.2 SELEX
In the 2'NH.sub.2 SELEXes, two alternate techniques were used to partition unbound RNA from sPLA.sub.2 /RNA complexes: nitrocellulose filtration and sPLA.sub.2 immobilized on polyclonal anti-sPLA.sub.2 beads as outlined in Table I. The elution of RNA from immobilized sPLA.sub.2 /RNA complexes was based on the observation that the binding of many ligands to sPLA.sub.2 is calcium dependent and on the premise that free polyclonal antibody can compete off bound ligands.
The starting pools for both the 50N7 (SEQ ID NO.:4) and 50N9 (SEQ ID NO.:7) SELEXes contained approximately 5.times.10.sup.14 RNA molecules (500 pmol) and bound sPLA.sub.2 with K.sub.d's of 71 and 48 nM, respectively. After 11 rounds, the 50N9 SELEX pool bound sPLA.sub.2 biphasically; the high affinity species which represented about 67% of the molecules, bound with a K.sub.d of 1.3 nM. Only a marginal improvement in affinity (K.sub.d =17 nM) was observed for the twelfth round 50N7 SELEX. Eleventh and twelfth round cDNAs from the 50N9 and 50N7 SELEXes, respectively, were PCR amplified with primers containing restriction sites and cloned into pUC18; 40 clones from the 50N9 and 41 from the 50N7 SELEX were sequenced. Sequences were aligned manually and are shown in Table II. The sequences from the 50N9 RNA SELEX are designated by ligand names including NN (SEQ ID NOs.:60-80) and the sequences from the 50N7 RNA SELEX are designated by ligand names including NS (SEQ ID NOs.:81-96).
C. RNA Sequences
Sequences identified by the sPLA.sub.2 SELEX procedures described above are shown in Table II (SEQ ID NOs.:10-96). In the 2'OH SELEXes, 16 of 51 (Standard SELEX) and 14 of 34 (Competition SELEX) sequenced ligands were unique. A unique sequence is operationally defined as one that differs from all others by three or more nucleotides. In Table II, the RNA sequences of all 2'OH RNA ligands are shown in standard single letter code (Cornish-Bowden (1985) Nuc. Acid Res. 13: 3021-3030). These clones fall into five sequence families (I-V) and a group of unrelated sequences (Orphans); ligands in all six groups bind with high affinity. In addition, 12 of 38 (50N9) and 12 of 40 (50N7) sequenced ligands were unique (Table II). All of the 50N9 ligands that bound sPLA.sub.2 with high affinity constituted a single sequence family (VI) which is the dominant family (19 of 40) of the 50N9 SELEX. Ligand NS2 which was isolated repeatedly from the 50N7 SELEX and the 2'OH Standard SELEX orphan 60 are related to family VI.
The data in Table II define consensus sequences for families I, II, III and VI (SEQ ID NOs.:97-100). The juxtaposition of the conserved sequences of family VI to the 5' fixed region and the conservation of the AGA by ligand NN2, suggest that these ligands require at least 3 nucleotides of the 5' fixed sequence for high affinity binding. Similarly, the family II alignment suggests that a 5' proximal CUC is necessary for high affinity binding; in some ligands this is a 5' fixed sequence, while in others it is an evolved sequence. Based on the juxtaposition of the conserved sequence of family I and II to the 3' fixed region an analogous logic predicts 3' boundaries within the 3' fixed region for ligands of these families.
The data in Table II also show that the distribution of sequence families I-V and orphans is different in the Standard and Competition SELEXes. Family I and V ligands occur frequently (18/51 and 9/51, respectively) in the Standard SELEX but are undetectable (0/34) in the Competition SELEX. A simple explanation for this difference is based on the observation that the high affinity binding of family I and V ligands and orphan 60 (and presumably the other orphans), unlike ligands of families II, III and IV, is calcium dependent. This correlation suggests that the free calcium was titrated by the high concentration of competitor tRNA, thus disallowing enrichment for high affinity, calcium dependent ligands.
D. Affinities
The dissociation constants for representative members of families I-VI, orphan and other ligands were determined by nitrocellulose filter binding experiments and are listed in Table III. Unlike random RNA, all tested ligands from the 2'OH SELEXes bound biphasically. Since the affinity determinations are made under conditions of protein excess, biphasic binding suggests that the ligand exists as two affinity species that are not in equilibrium, presumably these correspond to alternatively folded conformations. In most cases, the affinity of the low affinity species resembles that of random RNA which suggests that one folded conformation binds with high affinity and that all others bind like random RNA. Most high affinity species have dissociation constants from 0.2 to 2 nM which is 1200 to 120-fold improvement over random RNA.
Unlike the 2'OH ligands, the high affinity 2'NH.sub.2 ligands bind monophasically. The lone exception is family VI ligand NN27 (SEQ ID NO:61) which differs from the consensus sequence in having a rigorously conserved G replaced by a U. The affinity of family VI ligands is approximately 1 nM which is about a 50-fold improvement over that of random 2'NH.sub.2 RNA. The binding characteristics of the 2'OH and 2'NH.sub.2 ligands demonstrate that a priori it is not possible to know if a biphasic population is a collection of biphasic binders or a mixed population of high and low affinity ligands.
EXAMPLE 3
Ligand Truncation
To determine the minimal sequence necessary for high-affinity binding to sPLA.sub.2, boundary analyses were performed on representative members of Family I (ligand 2 (SEQ ID NO.:11)), Family II (ligands 72 and 721 (SEQ ID NOs.:20 and 33)), Family V (ligands 80 and 87 (SEQ ID NOs.:53 and 54)), Family VI (ligands NN41, NN11 and NN19 (SEQ ID NOs.:68, 60, and 64, respectively). Data for truncation/boundary determinations are shown in Table II (SEQ ID NOs.:101-121). The 3' boundaries of family I and II ligands are located in the 3' fixed region as is that of family V ligands. The position of the 5' boundary of family II ligands is in the 5' fixed region when the conserved CUC is fixed sequence. Similarly, the 5' boundary of family VI ligands is also in the 5' fixed region, while the 5' boundary of ligand 80 is the 5' G of the 5' fixed region. The 3' boundary of family VI ligands coincides with the 3' end of the consensus sequence.
Boundary locations were checked by determining the affinities of truncated ligands that approximate the minimal ligands. Although in general such truncates frequently bind as well as full length ligands, it is not a necessary outcome for at least two reasons. First, the boundary is primarily defined from the difference in affinity of the boundary species and the species that is one nucleotide shorter. Second, boundary experiments examine the affinity of ligands truncated on only one end, while the minimal ligand is truncated from both ends.
Binding affinities for full length and truncated ligands are shown in Table III. The truncates of ligands 2 and 87 (ligands 2.3 and 87.3, (SEQ ID NOs.:102 and 109) respectively) bind as well as their full length ligands, while truncates of 72, 72t and NN19 (72.c, 72t.2c and NN19.4, (SEQ ID NOs.:105, 106, and 117) exhibit a 5 to 10 fold loss in affinity. Also, the affinity of NN19.4 is restored to nearly full length levels by the addition of as few as six nucleotides to its 3' end (NN19.11, NN19.13, NN19.15 (SEQ ID NOs.:118, 119, and 120). While the low affinity of truncate 87.4 (SEQ ID NO.:110) confirms that the 5' G of ligands 80 and 87 is essential for high affinity binding, the high affinity of truncate 87.5 (SEQ ID NO:111) shows that the entire 5' fixed sequence is not necessary. The minimal ligand, operationally defined by the traditional boundary experiment, may include sequences that are required for an alternate function (i.e.., proper folding; the ligands must renature in the course of the experiment) or for no function at all. In other words, ligands that are shorter than the minimal ligand may bind with high affinity.
EXAMPLE 4
Specificity of RNA Ligands to Human sPLA.sub.2
The affinity of sPLA.sub.2 ligands 2, 72, 80, 87, NN19 and NN19.15 (SEQ ID NOs.:11, 20, 53, 54, 64, and 117, respectively) for proteins other than sPLA.sub.2 was determined by nitrocellulose partitioning (Tables IV and V). Like sPLA.sub.2, hen egg white lysozyme, bFGF and elastase are small, highly cationic proteins. Bovine pancreatic PLA.sub.2 is an evolutionarily and structurally related enzyme. The data in Table IV show that the ligands are highly specific for sPLA.sub.2. Specificity is particularly well illustrated by the affinities for bovine pancreatic PLA.sub.2 which is 10.sup.4 -fold less than that for human sPLA.sub.2. These data show that in general, evolved RNA ligands to sPLA.sub.2 bind to other proteins with an affinity similar to that of random RNA.
EXAMPLE 5
2'F Modification of 2'OH High Affinity Ligands
It was of interest to determine if 2'OH RNA ligands converted to a nuclease resistant form by the incorporation of 2' modified pyrimidines retained affinity for the target protein. Five 2'OH high affinity ligands to sPLA.sub.2 (ligands 11tF, 72F, 73tF, 86tF and 87F (SEQ ID NOs.:37, 21, 32, 50 and 55, respectively) were transcribed with 2'F CTP and 2'UTP in place of CTP and UTP and their binding affinities determined. As shown in Table III, both qualitative and quantitative changes were observed in the binding characteristics of four ligands; whereas all five bound biphasically as 2'OH RNA, they bound monophasically as 2'F RNA and their affinity was only marginally better (2 to 5 fold) than that of the random 2'F RNA control, regardless of their 2'OH affinity. On the other hand, the binding and inhibition characteristics of ligand 87 were unchanged by 2'F modification. No degradation of this RNA was observed after incubation in 0.5.times. serum for 3 hours.
EXAMPLE 6
Secondary Structure of High Affinity Ligands
In favorable instances, comparative analysis of aligned sequences enables deduction of secondary structure and structure-function relationships. Nucleotides that covary according to Watson-Crick base pairing rules are apt to be paired. Sequences that vary in composition and particularly in length are considered to be unimportant for function, while highly conserved sequences are apt to be directly involved in function.
Comparative analysis of family VI sequences yields a hairpin structure with a highly conserved, asymmetrical internal loop. The terminal loop (T-loop) is variable in both length and sequence (except the first and last positions) and is not apt to be directly involved in binding. The I-loop divides the stem in two. The V-stem varies in length (3-5 nucleotide pairs) and sequence. Three of the 5 base pairs are confirmed by Watson-Crick covariation. The C-stem is absolutely conserved. In this structure, the I-loop, C-stem and single stranded tails are critical for binding.
The suggested structure for family III ligands is a two plane G-quartet with a closing double helix, the sequence of which is not conserved. Based on the limited data, the G-quartet loop sequences and a 5' single stranded sequence may be conserved.
Family II contains two very highly conserved sequences: CUUACRG and GCYGAG. Without exception, the R and Y exhibit Watson-Crick covariation which strongly suggests that they are base paired, leading to a core structure consisting of a conserved, bulged stem that has an unpaired G adjacent to the 3' end of the 3' half of the stem. This G may be a loop or a bulged nucleotide.
EXAMPLE 7
Inhibition of PLA2-Mediated Contractions
The ligands of the invention are able to selectively block PLA2-mediated contractions of guinea pig lung pleural strips. The procedure described by Snyder et al. (Journal of Pharmacology and Experimental Therapeutics (1992) 262: 1147-1153) was followed to test the ability of the ligands to inhibit PLA2-induced contraction. Ligand 19.15 (SEQ ID NO: 121) was tested in this assay and showed a dose dependent inhibition of contraction. The same ligand was tested for its ability to inhibit an arachadonic acid-mediated contraction and did not act differently than the control. This example demonstrates that the ligands of the invention specifically inhibit PLA2-mediated contraction.
EXAMPLE 8
Inhibition of the sPLA.sub.2 Enzymatic Activity
To directly test the ability of the RNA ligands to inhibit sPLA.sub.2 enzymatic activity the procedure described by Reynolds et al. (Anal. Biochem. (1992) 204: 190-197) was followed. Ligands NN11 (SEQ ID NO: 113), NN19 (SEQ ID NO: 114), NN19.11 (SEQ IDNO: 118), NN19.13 (SEQ IDNO: 119), NN19.14(SEQ ID NO: 120), NN19.15 (SEQ ID NO: 121), 87 (SEQ ID NO: 54), 87F (SEQ ID NO: 55), 87.7 (SEQ ID NO: 112), 721 (SEQ ID NO: 104), 861 (SEQ ID NO: 49) were tested in this assay and showed an inhibitory effect.
The inhibition and affinity data for ligands 87 and 87.7 suggest a ligand with two domains. The minimal ligand, defined by boundary analysis, corresponds to the high affinity binding domain. A second domain, appended to the binding domain, is responsible for inhibition. The inhibition function may not be sequence dependent.
TABLE I__________________________________________________________________________SELEX CONDITIONS__________________________________________________________________________Random RNASEQ ID NO: Name Sequence__________________________________________________________________________1 30N1 RNA 5' gggagcucagaauaaacgcucaa-30N-uucgacaugaggcccggauccggc 3'2 N1 5' Primer 5' ccgaagcttaatacgactctatagggagctcagaataaacgctcaa 3'3 N1 3' Primer 5' gccggatccgggcctcatgtcgaa 3'4 50N7 RNA* 5' gggaggacgaugcgg-50N-cagacgacucgcccga 3'5 N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3'6 N7 3' Primer 5' tcgggcgagtcgtcctg 3'7 50N9 RNA* 5' gggaaaagcgaaucauacacaaga-50N-gcuccgccagagaccaaccgagaa 3'8 N9 5' Primer 5' taatacgactcactatagggaaaagcgaatcatacacaaga 3'9 N9 3' Primer 5' ttctcggttggtctctggcggagc 3'__________________________________________________________________________2'OH SELEX CONDITIONS30N1 SELEXSELEX Round [sPLA.sub.2 ] [RNA] [tRNA]__________________________________________________________________________Control 1-14 240 nM 2.4 uM --Standard 1-5 240 nM 2.4 uM -- 6-12 12 nM 120 nM --Competition 1-3 240 nM 2.4 uM -- 4-14 240 nM 2.4 uM 0.5 mM__________________________________________________________________________2'NH.sub.2 SELEX CONDITIONS50N9 & 50N7 SELEXIMMOBILIZED sPLA.sub.2 PARTITIONING sPLA2 RNA TBSC AbRound (pmol) (pmol) Gel Vol Total Vol Eluted RNA Eluted RNA__________________________________________________________________________1 0.2-2 468 1 ul 0.5 ml 2.6% 1.9%2 0.2-2 100 1 ul 1.0 ml 3.0% 2.6%3 0.4-4 345 2 ul 1.0 ml 0.1% 0.03%4 0.4-4 540 2 ul 1.0 ml 0.05% 0.03%5 2-20 730 10 ul 1.0 ml 0.07% 0.03%8 2-20 90 10 ul 1.0 ml 1.0% 0.2%9 2-20 102 10 ul 0.5 ml 2.4% 0.3%__________________________________________________________________________NITROCELLULOSE PARTITIONINGRound [sPLA.sub.2 ] [RNA] Vol Net RNA bound__________________________________________________________________________6 25 nM 373 nM 1.5 ml 2.1%7 10 nM 100 nM 1.5 ml 2.1%10 5 nM 50 nM 1.5 ml 3.8%11 5 nM 50 nM 1.5 ml 7.0%__________________________________________________________________________ *All C and U have 2'NH.sub.2 substituted for 2'OH for ribose
TABLE II - .cndot. Ligands Family I 10 71 gggagcucagaauaaacgcucaa------UCUCAU--GCUCGUCGCACGGCGUAACCUAUuucgac augaggcccggauccggc 11 2(6) gggagcucagaauaaacgcucaa------UCUCAUUGCUCGUCGCACGGCGUAACCUAUuucga caugaggcccggauccggc 12 3 gggagcucagaauaaacgcucaa-----AGCUCAUCGUC--UCGCAAGGCGUAUCCUAUuucgacau gaggcccggauccggc 13 77 gggagcucagaauaaacgcucaaACCUCGCCU-AU-GU---UCGCGCGGCGUAUCCUAu-ucgaca ugaggcccggauccggc 14 93(3) gggagcucagaauaaacgcucaa---CAGCCA-AU-GUGUCCCGUACGGCGUAUCCUAu-ucg acaugaggcccggauccggc 15 95 gggagcucagaauaaacgcucaa----CGCUGGAU-ACCA-UCGCACGGCGUAUCCUGCuucgaca ugaggcccggauccggc 16 85 gggagcucagaauaaacgcucaa--AUUGCAUCAU-GUA--CCGCAAGACGUAUUCUAu-ucgaca ugaggcccggauccggc 17 18 gggagcucagaauaaacgcucaa--AUCGCAUCAU-GUA--CCGCAAGACGUAUUCUAu-ucgaca ugaggcccggauccggc 18 54 gggagcucagaauaaacgcucaa--AUUGCAUCAU-GUA--CCGUAAGACGUAUUCUAu-ucgaca ugaggcccggauccggc 19 14 gggagcucagaauaaacgcucaa--AUUGCAUCAU-GUA--CCGCAAGACGUAUCCUAuuucgaca ugaggcccggauccggc 97 Consensus Sequence YMUCAU-GUH--YCGYAMGRCGUAUYCUAU Boundary Species 101 2 CUCAUUGCUCGUCGCACGGCGUAACCUAUuucgacauga Truncates and Derivatives 102 2.3 GG------UCUCAUUGCUCGUCGCACGGCGUAACCUAUuucgacaugag Family II 20 72(6) gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG------CCCG-----GCUGAG ACAC---------uucgacaugaggcccggauccggc 21 72F gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG------CCCG-----GCUGAGAC AC---------uucgacaugaggcccggauccggc 22 96(2) gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG------UUCG-----GCUGAG ACAC---------uucgacaugaggcccggauccggc 23 13t gggagcucagaauaaacgcucaaUGCCUCUG----CUUACGG------GUAAU----GCCGAGAC AC---------uucgacaugaggcccggauccggc 24 94 gggagcucagaauaaacgcucaaAGUCCUCUC----CUUACGG------UUCG-----CCCGAGAU AA---------uucgacaugaggcccggauccggc 25 15 gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG ------CCCG -----GCUGAGA CAU ---------uucgacaugaggcccggauccggc 26 79 gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG ------CUCG -----GCUGAGA CAC ---------uucgacaugaggcccggauccggc 27 88 gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG ------UCCG -----GCUGAGA CAC ---------uucgacaugaggcccggauccggc 28 81 gggagcucagaauaaacgcucaaGACCUCUG----CUUACAG ------CCCG -----GCUGAGA CGC ---------uucgacaugaggcccggauccggc 29 68t gggagcucagaauaaacgcucaaGGCCUCUG----CUUACGG ------CUAAU ----GCCGAG ACGC ---------uucgacaugpggcccggauccggc 30 75 gggagcucagaauaaacgcucaaGANCUCUG----CUUACAG ------CCCG -----GCUGGGA CAC ---------uucgacaugaggcccggauccggc 31 73t gggagcucagaauaaacgcucaa----CUUACAG ------UUCG -----GCUGAGAGAAGACG CAUACuucgacaugaggcccggauccggc 32 73tF gggagcucagaauaaacgcucaa----CUUACAG ------UUCG -----GCUGAGAGAAGAC GCAUACuucgacaugaggcccggauccggc 33 72t(2) gggagcucagaauaaacgcucaa----CUUACAG ------UUCG -----GCUGAGACGAA GAUCGACCuucgacaugaggcccggauccggc 34 71t(3) gggagcucagaauaaacgcucaa----CUUACAG--GAGAUUCCAUCUCGCUGAGACGC ---------uucgacaugaggcccggauccggc 35 1t gggagcucagaauaaacgcucaa----CUUACGGCAGCGAUUGCUG ---GCCGAGAAACC --------uucgacaugaggcccggauccggc 36 11t(2) gggagcucagaauaaacgcucaa----CUUACGG ------GUAAA ----GCCGAGAAAAU GUAUUGC-uucgacaugaggcccggauccggc 37 11tF gggagcucagaauaaacgcucaa----CUUACGG ------GUAAA ----GCCGAGAAAAUGU AUUGC-uucgacaugaggcccggauccggc 38 36t(9) gggagcucagaauaaacgcucaaUUGUCUUACAG------GUAAA ----GCUGAGGAAUCG UU -----uucgacaugaggcccggauccggc 39 4(6) gggagcucagaauaaacgcucaa-UGUCUUACGG------GUAAA ----GCCGAGAAAGUUUC C ----uucgacaugaggcccggauccggc 40 35t(3) gggagcucagaauaaacgcucaaGGCUGGG-UCUUUUACAG------GUAAA ----GCUGA GAA -----------uucgacaugaggcccggauccggc 41 89 gggagcucagaauaaacgcucaa-AGUCUUACGG------GUAAA ----GCCGAGAAAGUUUCC ----uucgacaugaggcccggauccggc 42 84t gggagcucagaauaaacgcucaaUCAUGUC-AUUACGG------GUAAA ----GCCGAGUUUC ---------uucgacaugaggcccggauccggc 43 79t gggagcucagaauaaacgcucaaAUCAUGUC-AUUACGG------GUAAA ----GCCGAGUUUC ---------uucgacaugaggcccggauccggc 98 Consensus Sequence CUCWR----CUUACRG ------BYMV -----GCYGAGA Boundary Species 103 72 aGACCUCUG----CUUACAG ------CCCG -----GCUGAGACAC ---------uucgacau gagg 104 72t uaaacgcucaa----CUUACAG ------UUCG -----GCUGAGACGAAGAUCGACCuucgac a Truncates and Derivatives 105 72.c GGGaaGACCUCUG----CUUACAG ------CCCG -----GCUGAGACAC ---------uu cgacaugaggcc 106 72t.2c GGGauaaacgcucaa----CUUACAG ------UUCG -----GCUGAGACGAAGAUCGAC Cuucgacau Family III 44 34t(2) gggagcucagaauaaacgcucaaCACGAGGGUGGGUGGGUGGCCGAGCGCUUGuucgacaug aggcccggauccggc 45 9t gggagcucagaauaaacgcucaaCACGGGGGUGGGUGGGUGGCCGAGCGCUUGuucgacaugaggc ccggauccggc 46 6t gggagcucagaauaaacgcucaaUGCCUCAUGCCAAUGUGGGAGGGUGGGUGGuucgacaugaggc ccggauccggc 47 76t gggagcucagaauaaacgcucaaCGCCUCAUGCCAACGCGGGAGGGUGGGUGGuucgacaugagg cccggauccggc 48 83t gggagcucagaauaaacgcucaaCGCCUCAUGCCAACGUGGGAGCGUGGGUGGuucgacaugagg cccggauccggc 49 86t gggagcucagaauaaacgcucaaCGCCUCAUGCCAAUGCGGGAGGGUGGGUGGuucgacaugagg cccggauccggc 50 86tF gggagcucagaauaaacgcucaaCGCCUCAUGCCAAUGCGGGAGGGUGGGUGGuucgacaugag gcccggauccggc 99 Consensus Sequence MAYGNGGGWGGGUGGGUGG Family IV 51 2t gggagcucagaauaaacgcucaaUCCGGGAGCUGAAAAA-CAUGCCGUUAGCCGuucgacaugagg cccggauccggc 52 70t gggagcucagaauaaacgcucaaUCCGCGAGCUGAAAAA-CAUGCCGUUAGCCAuucgacaugag gcccggauccggc Family V 53 80(7) gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCAGUGUCCAGCAUCuucgacauga ggcccggauccggc 54 87(2) gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacauga ggcccggauccggc 55 87F gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacaugagg cccggauccggc Boundary Species 107 80 gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCAGUGUCCAGCAUCuucgac 108 87.3 gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucga c Truncates and Derivatives 109 87.3 gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacaug 110 87.4 ggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacaug 111 87.5 gggagcu----auaaacgc ---aGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacau g 112 87.7 gggagcucagaauaaacgcucaaGCUCUGGGAUGAUGCCCACUGUCCAGCAUCuucgacaugu GUAGCUAAACAGCUUUAGGA Orphan Sequences 56 1 gggagcucagaauaaacgcucaaGCCGAACCGAAUGGAGGUGGAGGGAUUGCGuucgacaugaggcc cggauccggc 57 84 gggagcucagaauaaacgcucaaGACCACGUCCGAACGAACACCGCCACGCAuucgacaugaggcc cggauccggc 58 90 gggagcucagaauaaacgcucaaCCAACGACACUCACGCAUUGCCCACGAACGuucgacaugaggc ccggauccggc 59 60 gggagcucagaauaaacgcucaaACAACGGCCCACACGGGAGAUCCGAGAAAAGuucgacaugagg cccggauccggc Family V1 60 NN11(5) gggaaaagcgaaucauacacaagaCCGGCCGGGGAAA ---CCCGAGGUCCGAGGUAACGC AUGGCGCCUCACCGAGUCgcuccgccagagaccaaccgagaa 61 NN27 gggaaaagcgaaucauacacaagaCCGGCCGGGGAAA---CCCGAGGUCCGAUGUAACGCAUGG CGCCUCACCGAGUCgcuccgccagagaccaaccgagaa 62 NN16 gggaaaagcgaaucauacacaagaCCGGCCGGGGAAA---CCCGAGAUCCGAGGUAACGCAUGG CGCCUCACCGAGUCgcuccgccagagaccaaccgagaa 63 NN10 gggaaaagcgaaucauacacaagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUUGUACGAUG ACAACUCAGUgcuccgccagagaccaaccgagaa 64 NN19 gggaaaagcgaaucauacacaagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUGAACGA UAACUCGGUgcuccgccagagaccaaccgagaa 65 NN2(4) gggaaaagcgaaucauacacaagaGACCGGCCAGCCAAGGC-GCUGAGAUCCGAGGUUUCAG AACCCAUCGGGUUGgcuccgccagagaccaaccgagaa 66 NN11d gggaaaagcgaaucauacacaagaCCGGCCCGGUAUGUAGCCGGAGAUCCGAGACUUGCUGAA CGAGGUGCCACGgcuccgccagagaccaaccgagaa 67 NN29 gggaaaagcgaaucauacacaagaCCGGCCCGGUGUGUAGCCGGAGAUCCGAGACUUGCUGAAC GAGGUGCCACGgcuccgccagagaccaaccgagaa 68 NN41 gggaaaagcgaaucauacacaagaCCGGCCCGGUGUGCAGCCGGAGAUCCGAGACUUGCUGAAC AAGGUGCCACGgcuccgccagagaccaaccgagaa 69 NN5(2) gggaaaagcgaaucauacacaagaCCGGCCCCGCCAAUCAAGGGAGAUCCGAGGAAUUGGAA UGUUUGUGAGUGAgcuccgccagagaccaaccgagaa 100 Consensus Sequence ragaCCGGCCNGSNNNNNNNSCNGAGAUCCGAGG Boundary Species 113 NN11 aagaCCGGCCGGGGAAA ---CCCGAGGUCCGAGGUAACGCA 114 NN19 aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUG 115 NN2 gaGACCGGCCAGCCAAGGC-GCUGAGAUCCGAGGUUUCA 116 NN41 aagaCCGGCCCGGUGUGCAGCCGGAGAUCCGAGACUUGCUG Truncates and Derivatives 117 NN19.4 gggaaaag ------------aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUGA 118 NN19.11 gggaaaag ------------aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUG AACGAU --------------------agaccaaccgagaa 119 NN19.13 gggaaaag ------------aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUG --------------------------agaccaaccgagaa 120 NN19.14 gggaaaag ------------aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUG AACGAU ---------------------------ccgagaa 121 NN19.15 gggaaaag------------aagaCCGGCCGGCGCCAUA-GCCGAGAUCCGAGGUGUUG- --------------------------------ccgagaa 122 NS2(9) gggaggacgaugcggUAGUAACGGA ----UACUGAUCcGAGGUUAUAGUCACUAUAUCAU CCGCUGCGCcagacgacucgcccga Other Sequences 70 NN3 gggaaaagcgaaccauacacaagaGUGUGCGGUGCAUGCGUGGUGAAAGGGGGGUGGGGAAGAAA AACCGGCCCgcuccgccagagaccaaccgagaaa 71 NN4(9) gggaaaagcgaaucauacacaagaGUGUGCGGUGCAUGCGUGGUGAAAGGUGGGUUGUGGAG GAAGACCGUGCCgcuccgccagagaccaaccgagga 72 NN1 gggaaaagcgaaucauacacaagaCGGCGAGCAUGCGGCGAGUGGAGGGGGACUGAUGGAGGGCG AGACCGUGUgcuccgccagagaccaaccgagaa 73 NN6 gggaaaagcgaaucauacacaagaCGGCGAGCAGGCGGCGAGUGGAGGAGGACUGAUGGAGGGCG AGACCGCAGgcuccgccagagaccaaccgagaa 74 NN7 gggaaaagcgaaucauacacaagaCGGCGAGCAAGCGGCGAGUGGAGGAGGACUGAUGGAGGGCG AGACCGCAAgcuccgccagagaccaaccgagaa 75 NN22 gggaaaagcgaaucauacacaagaCGGCGAGCAUGCGGCGAGUGGAGGAGGACUGAUGGAGGGC GAGACCGUGUgcuccgccagagaccaaccgagaa 76 NN45 gggaaaagcgaaucauacacaagaCGGCGAGCAGGCGGCGAGUGGAGGAGGACUGAUGGAGGGC GAGACCGCGUgcuccgccagagaccaaccgagaa 77 NN40(2) gggaaaagcgaaucauacacaagaCCCCUUGAGCUCGUGACGCAGGAGGAGGGCCGAGGAG GAAAGUCGUCACAgcuccgccagagaccaaccgagaa 78 NN18 gggaaaagcgaaucauacacaagaGGAUGGCGGCAAGGCGCGAAAGGGAGGAUCGAGGAGGAAU CGCGUCAGGAgcuccgccagagaccaaccgagaa 79 NN30 gggaaaagcgaaucauacacaagaGCCAGCGAGUGUCGACAGUGUGGGUGGAAGUGACGGGAGG AUUGGAGACgcuccgccagagaccaaccgagaa 80 NN39 gggaaaagcgaaucauacacaagaCUGGUUGUGCGGACCCAGUGAGUGGGAGGACGUGAGGGUG GCAGCUGGgcuccgccagagaccaaccgagaa 81 NS20(17) gggaggacgaugcggAGGGUGGAUCGUGGAGGAAAAGCAUCGUGUGUAACCGAACCGAUC GUGGYcagacgacucgcccga 82 NS39(3) gggaggacgaugcggGUAGGGAUGAAGUGCGAUGUGAAUCCGGGUGCUAGCGAUGAUGUGU GCCCcagacgacucgcccga 83 NS38(2) gggaggacgaugcggGUAGGGAGACAGACACACACGCGGAAAGUAGAGCCAUCGUAACAUG CCCCcagacgacucgcccga 84 NS4 gggaggacgaugcggGUAGGGAUAAGCGAGUGUACAGCGAAUACGACUCGGAAUGCUUGGUGCGC cagacgacucgcccga 85 NS12(2) gggaggacgaugcggUGAAAGAGAAAGGUUGAGAUGAUUACAAGCGAAUUGGAUAAGUGUC UGGCcagacgacucgcccga 86 NS27 gggaggacgaugcggUGAAAUGAGAAAUGGAUUGAUGAUGAUUAcaagagAAUUGGAUAAGUGU CUGGUcagacgacucgcccga 87 NS11(2) gggaggacgaugcggGAGGGAAGGGUGGAACGGAACUCCGAUAAAGCUGUACAAGUACGUG GGGUcagacgacucguccga 88 NS1 gggaggacgaugcggAUAAGGAGGAGCAAGCGAGAAAUUGAGAAGUAACAAGAUCGACAUGGCCC cagacgacucgcccga 89 NS49 gggaggacgaugcggAUAAGGAUAAGAUCGAACGAGAGUGAACAAAGUUAAAUACAGUCUGGGG Gcagacgacucgcccga 90 NS48 gggaggacgaugcggGCUAAGGGAAGACAAUGAGAUAGCAGACAAUCAACUACACCCAUGUGCG Ucagacgacucgcccga 91 NS13 gggaggacgaugcggUGAGCUUAGGAUAGGAGCAACAAGUAGAGUAGAGUGAUAACUAGGGUGG Ccagacgacucgcccga 92 NS10 gggaggacgaugcggUGACAAAUGAGCAAGUAGCGAUAGAUGUGAUGGACAGAGACAGCCGGGG Ccagacgacucgcccga 93 NS14 gggaggacgaugcggAAAUGUGUUAGUGAAUGAUUGAGAGAAGAUAGAUGAUGUUGAAGUCUGG Ccagacgacucgcccga 94 NS6 gggaggacgaugcggAACuAAAAGACAGAGAGAAAACGACAAuACGAAGuACAuAuAACCCuGGC cagacgacucgcccga 95 NS18 gggaggacgaugcggAAGUAGAUGAUGGAUUGAGAUGUAAGUGUCAGUAUGAAGAGUCUCUGGG Ccagacgacucgcccga 96 NS7 gggaggacgaugcggAGGAAAUGAAGUAGUGAGAGUAUAACAUGAUUAUGAAUACGUGAUGUGGC cagacgacucgcccga *Fixed sequences are represented by lower case lettering; evolved sequenc by upper case. "-" indicates spacing for alignment. Ligands recovered fro the 2'OH Competition SELEX are denoted by "t"; F indicates 2'OH ligands which contain 2'F CTP and 2'F UTP in place of CTP and UTP, respectively. Sequences that were isolated more than once, are indicated by the parenthetical number, (n), following the ligand isolate number. .cndot.Sequence ID Number
TABLE III______________________________________Ligand SEQ ID NO Kd1 Kd2 Mole fraction______________________________________A: 2'OH LigandsFamily I71 10 1.3 nM 82 nM 0.602 11 1.7 nM 180 nm 0.452.3 102 2.6 nM 740 nM 0.553 12 1.9 nM 33 nM 0.5585 16 3.8 nM 92 nM 0.6093 14 17.2 nM 82 nM 0.5095 15 4.5 nM 140 nM 0.6518 17 5.0 nM 240 nM 0.5054 18 16.6 nM 350 nM 0.70Family II72 103 0.5 nM 270 nM 0.1372F 21 5.6 nM72c 104 4.6 nM 170 nM 0.1596 22 0.9 nM 130 nM 0.4079 26 1.7 nM 52 nM 0.3573t 31 2.4 nM 530 nM 0.4073tF 32 14.0 nM75t 30 0.9 nM 420 nM 0.4072t.2c 106 5.0 nM 360 nM 0.4011t 36 0.6 nM 110 nM 0.4011tF 37 7.0 nM4 39 0.7 nM 300 nM 0.5589 41 2.5 nM 62 nM 0.5035t 40 2.4 nM 170 nM 0.65Family III9t 45 39.4 nM 310 nM 0.256t 46 28.0 nM 1830 nM 0.5083t 48 10.5 nM 170 nM 0.3086t 49 2.8 nM 450 nM 0.3586tF 50 12.0 nMFamily V80 53 1.6 nM 300 nM 0.4087 54 0.8 nM 450 nM 0.5087F 55 1.2 nM 840 nM 0.5087.3 109 0.8 nM 720 nM 0.4087.4 110 4350 nM87.5 111 3.1 nM 1320 nM 0.5087.7 112 0.8 nM 250 nM 0.30Unrelated Sequence1 56 1.4 nM 43 nM 0.2560 59 0.3 nM 200 nM 0.20Random RNA30N1 1 360 nM30N1F 1 35 nMB: 2'NH.sub.2 LigandsFamily VINN11 60 2.8 nMNN27 61 0.4 nM 150 nM 0.2NN16 62 0.9 nmNN10 63 0.4 nMNN19 64 0.4 nMNN2 65 1.2 nMNN11d 66NN29 67 2.5 nMNN41 68 1.2 nMNN5 69 4.6 nMNN19.4 117 7.3 nMNN19.11 118 0.5 nMNN19.13 119 2.2 nMNN19.14 120 1.0 nMNN19.15 121 1.7 nMNS2 122 22.0 nMOther SequencesNN1 72 21 nMNN4 71 27 nMNN18 78 71 nMNN22 75 36 nMNN30 79 23 nMNN39 80 52 nMNN40 77 31 nMNS1 88 42 nMNS4 84 24 nMNS6 94 28 nMA: 2'NH.sub.2 LigandsNS7 96 18 nMNS10 92 22 nMNS11 87 16 nMNS12 85 40 nMNS13 91 15 nMNS14 93 12 nMNS18 95 52 nMNS39 82 16 nMNS48 90 42 nMNS49 89 26 nMRandom 2'NH.sub.2 RNA50N7 RNA 4 71 nM50N9 RNA 7 48 nM______________________________________
TABLE IV______________________________________Selectivity of sPLA.sub.2 RNA LigandsK.sub.d (nM) to specified protein*Seq. ID # Ligand Lysozyme bFGF bpPLA2 sPLA.sub.2______________________________________11 2 1,700 230 20,000 1.720 72 1,500 180 29,000 0.553 80 2,700 240 29,000 1.654 87 2,300 220 20,000 0.8 1 30N1 1,650 270 22,500 360______________________________________ *Standard binding experiments were performed to the specified target protein in TBSC and K.sub.d 's is calculated by curve fitting. The ligand bind no better than randomized RNA (30N1) to lysozyme (hen eggwhite), human basic fibroblast growth factor basic (bFGF) and bovine pancreatic PLA.sub.2. The sPLA.sub.2 K.sub.d that of the high affinity species.
TABLE V______________________________________Selectivity of sPLA.sub.2 2'NH.sub.2 RNA LigandsK.sub.d (nM) to specified protein*Seq. ID # Ligand Elastase bFGF Clq IgG sPLA.sub.2______________________________________ 64 NN19 40 210 -- >5,000 1121 NN19.15 40 165 1,000 4,300 0.4 7 50N9 70 170 1,000 >5,000 48______________________________________ *Standard binding experiments were performed to the specified target protein in TBSC and K.sub.d 's calculated by curve fitting. The ligands bind no better than randomized RNA (30N1) to human neutrophil elastase, human basic fibroblast growth factor (bFGF), and human immunoglobulin G (IgG and Clq).
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 122(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGGAGCUCAGAAUAAACGCUCAANNNNNNNNNNNNNNNNNNNNNNNNNNN50NNNUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 46 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:CCGAAGCTTAATACGACTCTATAGGGAGCTCAGAATAAACGCTCAA46(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GCCGGATCCGGGCCTCATGTCGAA24(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GGGAGGACGAUGCGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN50NNNNNNNNNNNNNNNCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:TAATACGACTCACTATAGGGAGGACGATGCGG32(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 17 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:TCGGGCGAGTCGTCCTG17(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GGGAAAAGCGAAUCAUACACAAGANNNNNNNNNNNNNNNNNNNNNNNNNN50NNNNNNNNNNNNNNNNNNNNNNNNGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:TAATACGACTCACTATAGGGAAAAGCGAATCATACACAAGA41(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:TTCTCGGTTGGTCTCTGGCGGAGC24(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:GGGAGCUCAGAAUAAACGCUCAAUCUCAUGCUCGUCGCACGGCGUAACCU50AUUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GGGAGCUCAGAAUAAACGCUCAAUCUCAUUGCUCGUCGCACGGCGUAACC50UAUUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GGGAGCUCAGAAUAAACGCUCAAAGCUCAUCGUCUCGCAAGGCGUAUCCU50AUUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GGGAGCUCAGAAUAAACGCUCAAACCUCGCCUAUGUUCGCGCGGCGUAUC50CUAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GGGAGCUCAGAAUAAACGCUCAACAGCCAAUGUGUCCCGUACGGCGUAUC50CUAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GGGAGCUCAGAAUAAACGCUCAACGCUGGAUACCAUCGCACGGCGUAUCC50UGCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:GGGAGCUCAGAAUAAACGCUCAAAUUGCAUCAUGUACCGCAAGACGUAUU50CUAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GGGAGCUCAGAAUAAACGCUCAAAUCGCAUCAUGUACCGCAAGACGUAUU50CUAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GGGAGCUCAGAAUAAACGCUCAAAUUGCAUCAUGUACCGUAAGACGUAUU50CUAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 78 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GGGAGCUCAGAAUAAACGCUCAAAUUGCAUCAUGUACCGCAAGACGUAUC50CUAUUUCGACAUGAGGCCCGGAUCCGGC78(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGCCCGGCUGAGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-F cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-F uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGCCCGGCUGAGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGUUCGGCUGAGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:GGGAGCUCAGAAUAAACGCUCAAUGCCUCUGCUUACGGGUAAUGCCGAGA50CACUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GGGAGCUCAGAAUAAACGCUCAAAGUCCUCUCCUUACGGUUCGCCCGAGA50UAAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGCCCGGCUGAGAC50AUUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGCUCGGCUGAGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGUCCGGCUGAGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:GGGAGCUCAGAAUAAACGCUCAAGACCUCUGCUUACAGCCCGGCUGAGAC50GCUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:GGGAGCUCAGAAUAAACGCUCAAGGCCUCUGCUUACGGCUAAUGCCGAGA50CGCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:GGGAGCUCAGAAUAAACGCUCAAGANCUCUGCUUACAGCCCGGCUGGGAC50ACUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:GGGAGCUCAGAAUAAACGCUCAACUUACAGUUCGGCUGAGAGAAGACGCA50UACUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-F cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-F uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:GGGAGCUCAGAAUAAACGCUCAACUUACAGUUCGGCUGAGAGAAGACGCA50UACUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:GGGAGCUCAGAAUAAACGCUCAACUUACAGUUCGGCUGAGACGAAGAUCG50ACCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:GGGAGCUCAGAAUAAACGCUCAACUUACAGGAGAUUCCAUCUCGCUGAGA50CGCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:GGGAGCUCAGAAUAAACGCUCAACUUACGGCAGCGAUUGCUGGCCGAGAA50ACCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:GGGAGCUCAGAAUAAACGCUCAACUUACGGGUAAAGCCGAGAAAAUGUAU50UGCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-F cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-F uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:GGGAGCUCAGAAUAAACGCUCAACUUACGGGUAAAGCCGAGAAAAUGUAU50UGCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:GGGAGCUCAGAAUAAACGCUCAAUUGUCUUACAGGUAAAGCUGAGGAAUC50GUUUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:GGGAGCUCAGAAUAAACGCUCAAUGUCUUACGGGUAAAGCCGAGAAAGUU50UCCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:GGGAGCUCAGAAUAAACGCUCAAGGCUGGGUCUUUUACAGGUAAAGCUGA50GAAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO: 41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:GGGAGCUCAGAAUAAACGCUCAAAGUCUUACGGGUAAAGCCGAGAAAGUU50UCCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:GGGAGCUCAGAAUAAACGCUCAAUCAUGUCAUUACGGGUAAAGCCGAGUU50UCUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:GGGAGCUCAGAAUAAACGCUCAAAUCAUGUCAUUACGGGUAAAGCCGAGU50UUCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:GGGAGCUCAGAAUAAACGCUCAACACGAGGGUGGGUGGGUGGCCGAGCGC50UUGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:GGGAGCUCAGAAUAAACGCUCAACACGGGGGUGGGUGGGUGGCCGAGCGC50UUGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:GGGAGCUCAGAAUAAACGCUCAAUGCCUCAUGCCAAUGUGGGAGGGUGGG50UGGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:GGGAGCUCAGAAUAAACGCUCAACGCCUCAUGCCAACGCGGGAGGGUGGG50UGGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:GGGAGCUCAGAAUAAACGCUCAACGCCUCAUGCCAACGUGGGAGCGUGGG50UGGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:GGGAGCUCAGAAUAAACGCUCAACGCCUCAUGCCAAUGCGGGAGGGUGGG50UGGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-F cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-F uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:GGGAGCUCAGAAUAAACGCUCAACGCCUCAUGCCAAUGCGGGAGGGUGGG50UGGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:GGGAGCUCAGAAUAAACGCUCAAUCCGGGAGCUGAAAAACAUGCCGUUAG50CCGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:GGGAGCUCAGAAUAAACGCUCAAUCCGCGAGCUGAAAAACAUGCCGUUAG50CCAUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCAGUGUCCAGC50AUCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGC50AUCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-F cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-F uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGC50AUCUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:GGGAGCUCAGAAUAAACGCUCAAGCCGAACCGAAUGGAGGUGGAGGGAUU50GCGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 76 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:GGGAGCUCAGAAUAAACGCUCAAGACCACGUCCGAACGAACACCGCCACG50CAUUCGACAUGAGGCCCGGAUCCGGC76(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 77 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:GGGAGCUCAGAAUAAACGCUCAACCAACGACACUCACGCAUUGCCCACGA50ACGUUCGACAUGAGGCCCGGAUCCGGC77(2) INFORMATION FOR SEQ ID NO:59:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 78 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:GGGAGCUCAGAAUAAACGCUCAAACAACGGCCCACACGGGAGAUCCGAGA50AAAGUUCGACAUGAGGCCCGGAUCCGGC78(2) INFORMATION FOR SEQ ID NO:60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:GGGAAAAGCGAAUCAUACACAAGACCGGCCGGGGAAACCCGAGGUCCGAG50GUAACGCAUGGCGCCUCACCGAGUCGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:61:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:GGGAAAAGCGAAUCAUACACAAGACCGGCCGGGGAAACCCGAGGUCCGAU50GUAACGCAUGGCGCCUCACCGAGUCGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:62:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:GGGAAAAGCGAAUCAUACACAAGACCGGCCGGGGAAACCCGAGAUCCGAG50GUAACGCAUGGCGCCUCACCGAGUCGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 97 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:GGGAAAAGCGAAUCAUACACAAGACCGGCCGGCGCCAUAGCCGAGAUCCG50AGGUUGUACGAUGACAACUCAGUGCUCCGCCAGAGACCAACCGAGAA97(2) INFORMATION FOR SEQ ID NO:64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 96 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:GGGAAAAGCGAAUCAUACACAAGACCGGCCGGCGCCAUAGCCGAGAUCCG50AGGUGUUGAACGAUAACUCGGUGCUCCGCCAGAGACCAACCGAGAA96(2) INFORMATION FOR SEQ ID NO:65:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:GGGAAAAGCGAAUCAUACACAAGAGACCGGCCAGCCAAGGCGCUGAGAUC50CGAGGUUUCAGAACCCAUCGGGUUGGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:66:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:GGGAAAAGCGAAUCAUACACAAGACCGGCCCGGUAUGUAGCCGGAGAUCC50GAGACUUGCUGAACGAGGUGCCACGGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:GGGAAAAGCGAAUCAUACACAAGACCGGCCCGGUGUGUAGCCGGAGAUCC50GAGACUUGCUGAACGAGGUGCCACGGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:68:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:GGGAAAAGCGAAUCAUACACAAGACCGGCCCGGUGUGCAGCCGGAGAUCC50GAGACUUGCUGAACAAGGUGCCACGGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:69:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 99 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:GGGAAAAGCGAAUCAUACACAAGACCGGCCCCGCCAAUCAAGGGAGAUCC50GAGGAAUUGGAAUGUUUGUGAGUGAGCUCCGCCAGAGACCAACCGAGAA99(2) INFORMATION FOR SEQ ID NO:70:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:GGGAAAAGCGAAUCAUACACAAGAGUGUGCGGUGCAUGCGUGGUGAAAGG50GGGGUGGGGAAGAAAAACCGGCCCGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:71:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:GGGAAAAGCGAAUCAUACACAAGAGUGUGCGGUGCAUGCGUGGUGAAAGG50UGGGUUGUGGAGGAAGACCGUGCCGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:72:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:GGGAAAAGCGAAUCAUACACAAGACGGCGAGCAUGCGGCGAGUGGAGGGG50GACUGAUGGAGGGCGAGACCGUGUGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:73:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:GGGAAAAGCGAAUCAUACACAAGACGGCGAGCAGGCGGCGAGUGGAGGAG50GACUGAUGGAGGGCGAGACCGCAGGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:74:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:GGGAAAAGCGAAUCAUACACAAGACGGCGAGCAAGCGGCGAGUGGAGGAG50GACUGAUGGAGGGCGAGACCGCAAGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:75:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:GGGAAAAGCGAAUCAUACACAAGACGGCGAGCAUGCGGCGAGUGGAGGAG50GACUGAUGGAGGGCGAGACCGUGUGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:76:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:GGGAAAAGCGAAUCAUACACAAGACGGCGAGCAGGCGGCGAGUGGAGGAG50GACUGAUGGAGGGCGAGACCGCGUGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:77:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:GGGAAAAGCGAAUCAUACACAAGACCCCUUGAGCUCGUGACGCAGGAGGA50GGGCCGAGGAGGAAAGUCGUCACAGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:78:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 98 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:GGGAAAAGCGAAUCAUACACAAGAGGAUGGCGGCAAGGCGCGAAAGGGAG50GAUCGAGGAGGAAUCGCGUCAGGAGCUCCGCCAGAGACCAACCGAGAA98(2) INFORMATION FOR SEQ ID NO:79:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 97 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:GGGAAAAGCGAAUCAUACACAAGAGCCAGCGAGUGUCGACAGUGUGGGUG50GAAGUGACGGGAGGAUUGGAGACGCUCCGCCAGAGACCAACCGAGAA97(2) INFORMATION FOR SEQ ID NO:80:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 96 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:GGGAAAAGCGAAUCAUACACAAGACUGGUUGUGCGGACCCAGUGAGUGGG50AGGACGUGAGGGUGGCAGCUGGGCUCCGCCAGAGACCAACCGAGAA96(2) INFORMATION FOR SEQ ID NO:81:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:GGGAGGACGAUGCGGAGGGUGGAUCGUGGAGGAAAAGCAUCGUGUGUAAC50CGAACCGAUCGUGGYCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:82:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:GGGAGGACGAUGCGGGUAGGGAUGAAGUGCGAUGUGAAUCCGGGUGCUAG50CGAUGAUGUGUGCCCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:83:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:GGGAGGACGAUGCGGGUAGGGAGACAGACACACACGCGGAAAGUAGAGCC50AUCGUAACAUGCCCCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:84:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:GGGAGGACGAUGCGGGUAGGGAUAAGCGAGUGUACAGCGAAUACGACUCG50GAAUGCUUGGUGCGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:85:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:GGGAGGACGAUGCGGUGAAAGAGAAAGGUUGAGAUGAUUACAAGCGAAUU50GGAUAAGUGUCUGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:86:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 85 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:GGGAGGACGAUGCGGUGAAAUGAGAAAUGGAUUGAUGAUGAUUACAAGAG50AAUUGGAUAAGUGUCUGGUCAGACGACUCGCCCGA85(2) INFORMATION FOR SEQ ID NO:87:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:GGGAGGACGAUGCGGGAGGGAAGGGUGGAACGGAACUCCGAUAAAGCUGU50ACAAGUACGUGGGGUCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:88:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:GGGAGGACGAUGCGGAUAAGGAGGAGCAAGCGAGAAAUUGAGAAGUAACA50AGAUCGACAUGGCCCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:89:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:GGGAGGACGAUGCGGAUAAGGAUAAGAUCGAACGAGAGUGAACAAAGUUA50AAUACAGUCUGGGGGCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:90:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:GGGAGGACGAUGCGGGCUAAGGGAAGACAAUGAGAUAGCAGACAAUCAAC50UACACCCAUGUGCGUCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:91:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:GGGAGGACGAUGCGGUGAGCUUAGGAUAGGAGCAACAAGUAGAGUAGAGU50GAUAACUAGGGUGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:92:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:GGGAGGACGAUGCGGUGACAAAUGAGCAAGUAGCGAUAGAUGUGAUGGAC50AGAGACAGCCGGGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:93:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:GGGAGGACGAUGCGGAAAUGUGUUAGUGAAUGAUUGAGAGAAGAUAGAUG50AUGUUGAAGUCUGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:94:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:GGGAGGACGAUGCGGAACUAAAAGACAGAGAGAAAACGACAAUACGAAGU50ACAUAUAACCCUGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:95:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:GGGAGGACGAUGCGGAAGUAGAUGAUGGAUUGAGAUGUAAGUGUCAGUAU50GAAGAGUCUCUGGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:96:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:GGGAGGACGAUGCGGAGGAAAUGAAGUAGUGAGAGUAUAACAUGAUUAUG50AAUACGUGAUGUGGCCAGACGACUCGCCCGA81(2) INFORMATION FOR SEQ ID NO:97:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:YMUCAUGUHYCGYAMGRCGUAUYCUAU27(2) INFORMATION FOR SEQ ID NO:98:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:CUCWRCUUACRGBYMVGCYGAGA23(2) INFORMATION FOR SEQ ID NO:99:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 19 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:MAYGNGGGWGGGUGGGUGG19(2) INFORMATION FOR SEQ ID NO:100:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:RAGACCGGCCNGSNNNNNNNSCNGAGAUCCGAGG34(2) INFORMATION FOR SEQ ID NO:101:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:CUCAUUGCUCGUCGCACGGCGUAACCUAUUUCGACAUGA39(2) INFORMATION FOR SEQ ID NO:102:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:GGUCUCAUUGCUCGUCGCACGGCGUAACCUAUUUCGACAUGAG43(2) INFORMATION FOR SEQ ID NO:103:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 42 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:AGACCUCUGCUUACAGCCCGGCUGAGACACUUCGACAUGAGG42(2) INFORMATION FOR SEQ ID NO:104:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 48 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:UAAACGCUCAACUUACAGUUCGGCUGAGACGAAGAUCGACCUUCGACA48(2) INFORMATION FOR SEQ ID NO:105:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 48 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:GGGAAGACCUCUGCUUACAGCCCGGCUGAGACACUUCGACAUGAGGCC48(2) INFORMATION FOR SEQ ID NO:106:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 53 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:GGGAUAAACGCUCAACUUACAGUUCGGCUGAGACGAAGAUCGACCUUCGA50CAU53(2) INFORMATION FOR SEQ ID NO:107:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 59 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCAGUGUCCAGC50AUCUUCGAC59(2) INFORMATION FOR SEQ ID NO:108:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 59 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGC50AUCUUCGAC59(2) INFORMATION FOR SEQ ID NO:109:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 62 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGC50AUCUUCGACAUG62(2) INFORMATION FOR SEQ ID NO:110:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 61 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:GGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGCA50UCUUCGACAUG61(2) INFORMATION FOR SEQ ID NO:111:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 55 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:GGGAGCUAUAAACGCAGCUCUGGGAUGAUGCCCACUGUCCAGCAUCUUCG50ACAUG55(2) INFORMATION FOR SEQ ID NO:112:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 83 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:GGGAGCUCAGAAUAAACGCUCAAGCUCUGGGAUGAUGCCCACUGUCCAGC50AUCUUCGACAUGUGUAGCUAAACAGCUUUAGGA83(2) INFORMATION FOR SEQ ID NO:113:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:AAGACCGGCCGGGGAAACCCGAGGUCCGAGGUAACGCA38(2) INFORMATION FOR SEQ ID NO:114:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:AAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUG38(2) INFORMATION FOR SEQ ID NO:115:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:GAGACCGGCCAGCCAAGGCGCUGAGAUCCGAGGUUUCA38(2) INFORMATION FOR SEQ ID NO:116:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 41 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:AAGACCGGCCCGGUGUGCAGCCGGAGAUCCGAGACUUGCUG41(2) INFORMATION FOR SEQ ID NO:117:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 47 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:GGGAAAAGAAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUGA47(2) INFORMATION FOR SEQ ID NO:118:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 66 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:GGGAAAAGAAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUGAACG50AUAGACCAACCGAGAA66(2) INFORMATION FOR SEQ ID NO:119:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 60 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:GGGAAAAGAAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUGAGAC50CAACCGAGAA60(2) INFORMATION FOR SEQ ID NO:120:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 59 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:GGGAAAAGAAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUGAACG50AUCCGAGAA59(2) INFORMATION FOR SEQ ID NO:121:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 53 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:GGGAAAAGAAGACCGGCCGGCGCCAUAGCCGAGAUCCGAGGUGUUGCCGA50GAA53(2) INFORMATION FOR SEQ ID NO:122:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 81 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: ALL C'S ARE 2'-NH.sub.2 cytosine(ix) FEATURE:(D) OTHER INFORMATION: ALL U'S ARE 2'-NH.sub.2 uracil(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:GGGAGGACGAUGCGGUAGUAACGGAUACUGAUCCGAGGUUAUAGUCACUA50UAUCAUCCGCUGCGCCAGACGACUCGCCCGA81__________________________________________________________________________

	Number	Date	Country
Parent	714131	Jun 1991
Parent	536428	Jun 1990

High-affinity oligonucleotide ligands to secretory phospholipase A2 (sPLA.sub.2)

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