IE920561A1 - Aptamer specific for thrombin and methods of use - Google Patents

Abstract

Oligonucleotide sequences that mediate specific binding to thrombin and optionally contain modified bases, sugars, or sugar linkages are disclosed. Single-stranded DNA oligomers are obtained that bind thrombin and inhibit its function in vitro and in vivo. The thrombin binding oligomers are useful for therapeutic, diagnostic and manufacturing purposes. An improved method for identifying these oligomers is also described, involving complexation of the support-bound thrombin with a mixture of oligonucleotides containing random sequences under conditions wherein a complex is formed with the specifically binding sequences, but not with the other members of the oligonucleotide mixture. The thrombin-oligonucleotide complexes are then separated from both the support and the uncomplexed oligonucleotides and the complexed members of the oligonucleotide mixture are recovered from the separated complex and subsequently amplified using standard techniques.

Description

AMP MBTHQD8-QP USB Technical Field This invention ie in the field of rational drug design using biomolecule targeting and aptamer development. The invention diacloses and claims methods for making aptamers to thrombin and the aptamers resulting therefrom which may be applied broadly to diagnostics and therapeutic!. More specifically, thia invention is related to aptamera that bind to thrombin and interfere vith ita normal biological function, and therapeutic usee for these aptamers.

Background and Related Art Specifically Binding Oligonucleotides. Conventional methods of detection and isolation of proteins and other molecules have employed antibodies and the like which specifically bind such substances, Recently, however, ths dt fiflSCQ design of specifically binding oligonucleotides for non-oligonucleotide targets that generally bind nucleic acids has been deecribed. 8ee, e.g., Blackwell, T.K., et al., Selmca (1990) 2£QillO4xxxu? Blackwell, x.x., wu «X., ρι.ι·ιιι·ι· (x»»o) saa.xxx? 1152} Tuerk, C., and Sold, P., Science (1990) 241»505510} Joyce, O.P., O«ne (1989) £2i83-87. Such oligonucleotides have been termed ·aptamers* herein.

TUerk and Gold describe the use of an ia vitro selection and enrichment procedure. Xn this method, a pool of RNAs that are completely randomized at specific positions ie subjected to selection for binding by a desired nucleic acid-binding protein which is then bound to a nitrocellulose filter. The bound RNAs then are recovered and amplified ae double-stranded DNA that ie competent -3for subsequent la vitro transcription. The newly transcribed RNA then is recycled through this procedure to enrich for oligonucleotides that have coneeneua sequence! for binding by the cognate protein. The oligonucleotides so obtained then may be sequenced for further study. Tuerk and Gold applied this procedure to Identify RNA oligonucleotides which are bound by the RNA binding region of T4 DNA polymerase.

Kinsler, K.W., et al., Nucleic Acids Rea. (1989) 12*·3645-3653, applied this technique to Identify double-stranded DNA sequence! that were bound by proteine that bind to DNA and regulate gene expression, in the reported work, total genomic DMA ie firet converted to a form that ie suitable for amplification by PCR by lb llgacion of linker sequences to the yeuuudu SNA fragments and the DNA sequences of interest are selected by binding mediated by the target regulatory protein. The recovered bound sequences are then amplified by PCR. The proceea of binding by protein and amplification are repeated aa needed. The selection and amplification proceea are repeated ae needed. The proceea aa described was applied to identify DNA sequence! which bind to the Xenopua laevla transcription factor 3A. The earns authore (Kinsler et al.) in a later paper, Mol. Cell BloL· (1990) :634-842, applied thie eame technique to identify the portion of the human genome which is bound by the OLI gene product produced as a recombinant fusion protein.

The OLI gene is amplified in a subset of human tumors.

Bll ing ton, A.D., et al., Nature (1990) 818-822, describe the production of a large number of random sequence RNA molecules and identification of those which bind specifically to immobilised target molecules, in the case of thie paper, to specific dyes such as Cibacron blue. Randomly synthesised DNA yielding approximately 1015 individual sequences was amplified by -3PCR and transcribed into RNA. It waa thought that the coaq>lexity of Cha pool waa reduced in the amplificatioa/tranecription steps to approximately 1013 different eequencee. The pool wae then applied to an affinity column containing the dye and the bound sequence· eubeequently eluted, treated with reverse transcriptase and amplified by PCR. The results showed that about one in 1010 random sequence RNA molecules folds in such a way ae to bind specifically to the ligand.

Thiesen, H.-J., and Bach, C., Nucleic Acids &UU (1990) 11:3203-3200, describe what they call a target detection assay (TDA) to determine double-stranded DNA binding sites for putative DNA binding proteins. In their approach, a purified functionally active DNA binding protein and a pool of random double-stranded oligonucleotides which contain PCR primer sites at each end were incubated with the protein. The resulting DNA complexes with the protein (in their case, the SR-1 regulatory protein) were separated from the unbound oligomers in the random mixture by band-shift electrophoresis and the 6P-1 bound oligonucleotides were rescued by PCR and cloned, and then sequenced.

None of the cited references describe the use 2S of single-stranded DMA ae an appropriate material for generating aptamere. The uee of DNA aptamere hae several advantages over RNA ineluding increased nuclease stability (Shaw, J.P. et al., wue Aeld Res (1991) 11:747750), in particular plasma nuclease stability, and ease of amplification by PCR or other methods. RNA generally le converted to DMA prior to amplification using reverse transcriptase, a process that ie not equally efficient with all sequences, resulting in lose of some aptamere from a selected pool. -4Finally, non· of the above reference· describes (1) the identification of oligonucleotides which specifically bind to thrombin, which does not normally bind to DNA; (li) interference with the normal biological function of target molecules such as thrombin due to binding; (iii) the use of linkages other than the standard phosphodiester linkages in the backbone of the oligonucleotide, (iv) the use of base analogs in the oligonucleotide, (v) target-specific binding of short aptamer sequences and aptamer analog sequences derived from a larger full-length parent aptamer molecule, (vi) In vivo therapeutic efficacy of an aptamer or (vll) in vivo therapeutic efficacy of an aptamer analog.

Thrombin. Acute vascular diseases are associated with partial or total occlusion of a blood vessel by blood clots, which contain platelets and fibrin. These diseases Include serious health risks such as oiyocardial infarction, deep vain thrombosis, pulmonary embolism, peripheral arterial occlusion and the like.

Treatment or prophylaxis of thrombotic diseases is based on either inhibition of clotting or acceleration of thrombolysis. Both approaches to treatment of thrombotic disease have been deeeribed using agents such as heparin or hirudin to inhibit thrombin and streptokinase or tissue plasminogen activator to accelerate thrombolyeie. However, a need remains for improved therapeutic agente that inhibit the activities of thrombin in clot formation, platelet aggregation or activation and other thrombin-mediated processes.

Thrombin is a multifunctional enzyme that (1) converts fibrinogen to fibrin by enzymatic cleavage; (il) has mitogenic effects on lymphocytes and vascular smooth muscle cells; (iii) stimulates platelet aggregation and activation; (iv) is chemotactic for monocytes; (v) stimulates vascular endothelial cell mediated production -5of prostacyclin, platelet-activating factor and other factora/ (vi) induce· neutrophil adherence to veeeel wall·; (vii) stimulates vascular endothelial cell adhesion phenotype; and (viii) generates activated protein C by cleavage of proteia C.

Mitogenic activity of thrombin is exerted through binding to thrombin receptors (Coughlin, S.R., et al, J. Clin, Invest.. (1992) ££:351-355). Platelet aggregation, which plays a major role in arterial thrombosis is largely dependent on the function of thrombin (Hanson, 8.R., et al, Proc. Natl. Acad, flci.

USA. (1988) £1(3184-3188). Platelets carry functional thrombin receptors. Inflammatory responses can also bs mediated by thrombin through stimulation of platelet activating factor (PAF) (Preecott, 8., et al, Proc. Natl. Acad. Sci, USA. (1984) £1*3534-3538. FAF promotes adhesion of neutrophils to endothelial matrix, leading to degranulation of the neutrophils and an associated inflammatory response.

Disclosure of the Invention The identification of ollgonucleotidee that epeclfically bind to thrombin, which does not normally bind to RNA or DNA, has now been demonstrated. The thrombin aptamers bind to thrombin and inhibit both its catalytic activity in converting fibrinogen to fibrin and lte platelet aggregating activity. The aptamers are potent inhibitors of thrombin function and represent a new class of pharmaceutical agents for modulation of the activity of this protease. The molecules of this invention may be utilised in compositions and methods for Inhibiting any thrombin-mediated or thrombin-associated procaea or function. Pharmaceutical compositions containing these molecules, as well ae methods of treatment or prophylaxis of vascular diseases, -βinflammatory rssponses, cancer-related hypercoagulable states, sepsis and neural vasooclusive diseases using these compositions are also part of the present invention. These molecules can also be utilised in compositions and methods for la vitro or la vivo imaging, diagnosis, for storing and treating extracorporeal blood and for coating implant devices.

These molecules can be synthesised chemically or enzymatically as described below, and can be prepared in conmercial quantities. The aptamers of the present invention are composed of DNA and chemically related molecules. DNA is a class of molecule ordinarily found in animals and it is expected that the ismunogenieity of throotbin aptamers will be nonexistent or very low.

Immune reactions against nucleic acids are known to be rare and, when observed, are associated with autoimmune disorders. Because of their compatibility with biological systems, the molecules of the Invention are suitable in the treatment of both acute and nonacute vascular conditions.

Xn one aspect, the invention is directed to a method to determine an aptamer which binds specifically to thrombin, which method comprisea providing a mixture containing oligomers having portions which form a random set of sequences and portions which permit amplification of the oligomers, incubating the oligomer mixture with thrombin coupled to a support to form complexes between thrombin and the oligomers bound specifically thereto, removing the unbound members of the oligonucleotide mixture from the support environment, recovering the complexed oligonucleotide(s) from the support, anpllfying the recovered oligonucleotides, and asqusneing the recovered and amplified oligonucleotide(s) which had been complexed with thrombin, la a preferred embodiment, the mixture of oligonucleotides having random sequences also -7contains a consensus sequence known to bind to thrombin.

In another aspect, the invention is directed to a method to determine an aptamer which binds specifically to thrombin, which method comprises providing a mixture containing oligomers having portions which form a random set of sequences and, optionally, portions which permit salification of the oligomers, Incubating the oligomer mixture with thrombin coupled to a support to form complexes between thrombin and the oligamere bound specifically thereto, removing the unbound members of the oligonucleotide mixture from the support environment, recovering the complexed oligonucleotides by uncoupling the thrombin from the support, amplifying the recovered oligonucleotides, and sequencing the recovered and amplified oligonucleotides which had been complexed with the thrombin. In a preferred embodiment, the mixture ot oligonucleotides having random sequences also contains a sequence or sequences that permit binding to thrombin.

In a particularly preferred embodiment, the oligonucleotide mixture is single*stranded DMA.

In other aspects, the invention is directed to oligonucleotides which contain sequences identified by the above methods, and to oligonucleotide sequences which bind specifically to thrombin, in still another aspect, the Invention ie directed to complexes comprising the thrombin target substance and specifically bound oligomer.

In still other aspects, the invention is directed to oligomers which contain sequences that bind specifically to thrombin target substances and inhibit its normal biological function, and to the use of these oligomers in therapy, diagnostics, and purification procedures.

In yet a further aspect, this invention is directed to oligomers which contain sequences that bind -ιepecifically to throobin and inhibit· it· normal biological function, and which also contain one or more modified baeea, sugars, or sugar linkages, and to the uee of these oligomers in therapy, diagnostics, and purification procedures.

Brief Description of the Figures Figure 1 is a chart depicting thrombin aptamer consensus-related sequences.

Figure 2 is a plot of la vivo thrombin inhibition obtained from primates using a 15-mer aptamer.

Modes of Carrying Out the Invention The practice of the present invention encom15 passe· conventional techniques of chemistry, molecular biology, biochemistry, protein ehemistry, and recombinant DNA technology, which are within the skill of ths art. Such techniques are explained fully in the literature. \ flee, e.g., Oligonucleotide Synthesis (M.J. Gait ed. 1984)/ Nucleic Acid Hybridisation (B.D. Barnes t S.J.

Higgins, eds., 1984); Sambrook, Fritsch ft Maniatis, Molecular Cloning.;—A Laboratory Manual, Second Bdition (1989); FO Technology (H.A. Brlich ed., Stockton Frese); r.k. scope, Frgttia..Fu£iflcatloa Briadplifl,.and .Exact ica (Sprlnger-Verlag); and the serie· Method· in Baaymology (S. Colowick and N. Kaplan eds., Academic Frees, inc.).

All patents, patent applications and publications mentioned herein, whether supra or infra, are hereby incorporated by reference in their entirety.

The invention is directed to a method which permits the recovery and deduction or identification of aptamers which bind specifically to thrombin and compositions that result from ths use of the method.

For example, these aptamers can be used as a separation tool for retrieving or detecting thrombin. In -9these method·, the aptamers function much like monoclonal antibodies In their specif icity and usage. By coupling the aptamers containing the specifically binding sequences to e solid support, thrombin can bs rscovered in useful quantities. In addition, these aptamers can be used in diagnosis by employing them in specific binding assays.

For application in such various uses, the aptamers of the invention may be coupled to auxiliary substances that enhance or complement the function of the aptamer. Such auxiliary substances include, for example, labels such as radioisotopes, fluorescent labels, enzyme labile and the like; specific binding reagents such as antibodies, additional aptamer sequence, cell surface receptor ligands, receptor· per ee and the like; toxins euoh ae diphtheria toxin, tetanus toxin or ricin; drugs such as antiinflammatory, antibiotic, or metabolic regulator pharmaceuticals, solid supports such as chromatographic or electrophoretic supports, and the like. Suitable techniques for coupling of aptamers to desired auxiliary eubetances are generally known for a variety of such auxiliary substances, and the specific nature of the coupling procedure will depend on the nature of the auxiliary substance chosen. Coupling may be direct covalent coupling or may Involve the use of synthetic linkers such as those marketed by Pierce Chemical Co., Rockford, XL.

Aa used herein, specifically binding oligonucleotides or aptamers” refers to oligonucleotides having specific binding regions which are capable of forming complexes with thrombin in an environment wherein other substances in the same environment are not complexed to the oligonucleotide.

The specificity of the binding is defined in terms of the comparative dissociation constants of the aptamer for -10thrombin a· compared to the dissociation constant with respect to the aptamer and other materials in ths environment or unrelated molecules in general.

Typically, the Xd for the aptamer with respect to thrombin will be 10-fold less than Xd with respect to aptamer and the unrelated material or accoepanying material in the environment. Preferably the Xd will be 50-fold leas, more preferably 100-fold less, and more preferably 200-fold less.

As specificity is defined, in terms of Xd as set forth above, excluded from ths categories of unrelated materials and materials accompanying thrombin in ita environment are thoae materials which art sufficiently related to thrombin to be immunologlcally croesreactive therewith, and materials which natively bind oligonucleotides of particular sequences such as nucleases, restriction ensymes, and ths like. By inmunologically croesreactive is meant that antibodies raised vith respect to thrombin crossreact under standard assay conditions with the candidate material. Generally, for antibodies to crossreact in standard assays, ths binding affinities of the antibodies for croesreactive materials should be in the range of 10-fold.

Thus, aptamers which contain specific binding regions are specific vith respect to unrelated materials and with respect to materials which do not normally bind such oligonucleotides such as nucleases and restriction ensymes. Xn general, a minimum of approximately 6 nucleotides, preferably 10, and more preferably 14 nucleotides, are necessary to effect specific binding. Oligonucleotides of sequences as short as 6 bases have been shown to specifically bind and inhibit thrombin.

The only apparent limitations on the binding specificity of the thrcmhln/ollgonucleotlde couples of the Invention concern sufficient sequence to be distinctive in the -Ilbinding oligonucleotide and sufficient binding capacity of thrombin to obtain the necessary interaction. Oligonucleotides of eequencee shorter than 10, e.g., β mere, are feasible if the appropriate interaction can be S obtained m tne context or the environment in whiufe the thrombin ie placed. Thue, if there are fev interferences by other materials, less specificity and less strength of binding may be required.

Ae used herein, aptamer” refere in general to 10 either an oligonucleotide of a tingle defined sequence or a mixture ot eaid oligonucleotides, wherein the mixture retains the properties of binding specifically to thrombin. Thue, ae used herein aptamer denotes both singular and plural eequencee of oligonucleotides, as defined hereinabove.

Structurally, the aptamere of the invention are specifically binding oligonucleotides, wherein oligonucleotide ie ae defined herein. As set forth herein, oligonucleotides Include not only thoee with conventional bases, sugar residues and interaucleotlde linkages, but also thoee whloh contain modifications of any or all of these three moieties.

Oligomers or oligonucleotides” include RNA or ONA sequences of more than one nucleotide in either single chain or duplex form end specifically includes short eequencee such ae dimers and trimers, in either single chain or duplex font, which may be intermediates in the production of the specifically binding oligonueleotldee.

Oligonucleotide· or oligomer le generic to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forme thereof), i.e., ONA, to polyribonucleotides (containing D-rlboee or modified forme thereof), i.e., RNA, and to any other type of polynucleotide which is an N-glycoside or C-glycoside of s purine or f pyrimidine base, or modified purine or pyrimidine base.

The oligomers of the invention may be formed using conventional phosphodiester-linked nucleotides and S synthesized using standard solid phase (or solution phase) oligonucleotide synthesis techniques, which are now commercially available. However, the oligomers of the invention may also contain one or more substitute linkages as is generally understood in the art. Some of these substitute linkages are non-polar and contribute to the desired ability of the oligomer to diffuse across membranes. These substitute linkages are defined herein as conventional alternative linkages such as phosphorothioete or phosphoramidats, are synthesized as described in the generally available literature.

Alternative linking groups include, but are not limited to embodiments wherein a moiety of the formula P(O)8, (thioate), P(S)8 (dithioate), P(0)»»a, P(O)R', P(O)ORe, CO, or CONR'£, wherein R* is H (or a salt) or alkyl (1-120 and R* is alkyl (1-90 ia joined to adjacent nucleotides through -0- or -S-. Di thioate linkages are disclosed and claimed in commonly owned U.S. application no. 248,517. Substitute linkages that may be used in the oligomers disclosed herein also Include nonphosphorous-based internucleotide linkages such as the 3'-thioformacetal (-S-CHj-O·), formacetal (-O-CHj-O·) and 3*-amine (-NH-CHj-CHj-) lntsrnudeotide linkages disclosed and claimed in commonly owned pending U.S. patent application serial nos. 490,784 and 743,130, both incorporated herein by reference. One or more substitute linkages may be utilised in the oligomers in order to further facilitate binding with complementary target nucleic acid sequences or to increase the stability of the oligomers toward nucleases, as well as to confer -13permeation ability. (Not all such linkages in the earns * oligomer need be identical.) The term nucleoside or nucleotide ie eimilarly generic to ribonucleosides or ribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or to any other nucleoside which is an It-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base. Thus, the stereochemistry of the sugar carbons may be other than that of D-ribose in one or more residue!. Also included are analoge where the ribose or deoxyribose moiety is replaced by an alternate etructure such as the 6-membered morpholino ring described in U.S. patent number 5,034,506 or where an acyclic structure serves as a scaffold that positions the bass analogs described herein in a manner that permits efficient binding to target nucleic acid sequences or other targets, elements ordinarily found in oligomers, such as the furanose ring or the phoephodiester linkage may be replaced with any suitable functionally equivalent element. As the « ancmer binds to duplexes ia a manner similar to that for the 8 anomsrs, one or mors nucleotides may contain this linkage or a domain thereof.

(Praseuth, D., at al., Proc Natl Acad Sci (USA) (1988) 11:1349-1353). Modifications in the sugar moiety, for example, wherein one or mors of the hydroxyl groups ars replaced with halogen, aliphatic groups, or functionalised as ethers, amines, and the like, are also included. nucleoside and nucleotide include those moieties which contain not only the natively found purine and pyrimidine bases λ, T, C, O and U, but also modified or analogous forms thereof. Modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocyclee. Such analogous purines and analogous pyrimidines are those generally •14known in the art, many of which are uaad as chemotherapeutic agents. An exemplary but not exhaustive list includes pseudoisocytosine, M^^-ethanocycosine, 8hydroxy-N^-methyladeniae, 4-acetylcytosine, *55 (carboxyhydroxylmethyl) uracil, 5-fluorouracil, ·bromouracil, 5 - carboxymethylaminomethyl · 2 - thiouracil, -carboxymethylaminooethyl uracil, dihydrouracil, inoaine, N^-isopentenyl-adenine, l-methyladenine, l-methylpeeudouracil, l-methylguanine, l*methylinoeine, 2,2-dimethylguanine, 2-methyladenlne, 2-methylguanine, 3me thy Icy toe ine, 5-methyl cytosine, Ne-methyl adenine, 7methylguanine, 5 - me thylami nomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, beta-D-mannosylgueoeine, 5’methoxycarbonyImethyluracil, 5-methoxyuracil, 213 methylthio-N®-isopent enyl adenine, uradl-5-oxyacetic acid methyl ester, pseudouracil, 2-thiocytosine, 5-methyl-2thiouracll, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, queosine, 2-thiocytosine, and 2,6-diamlnopurine.

In addition to the modified bases above, nucleotide residues which are «basic, i.e., devoid of a purine or a pyrimidine base may aleo be included in the aptamers of the invention and in the methods for their obtention.

The sugar residues in the oligonucleotides of the invention may aleo be other than conventional ribose and deoxyribose residues. Xn particular, substitution at ths 2' -position of the furanoee residue ie particularly important.

Aptamer oligonucleotides may contain analogous forms of ribose or deoxyribose sugars that are generally known in the art. An exemplary, but not exhaustive list includes 2' substituted sugars such as 2'-0-methyl-, 2’0-allyl, 2' -fluoro- or 2*-a si do-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as · arabinose, xyloses or lyxoses, pyranose sugars, furasose sugars, sedoheptuloses, acyclic analoge and abaoic nucleoside analogs such as methyl riboside.

Although the conventional sugara and bases will be used in applying the method of the Invention, substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing the final product. Additional techniques, such as methods of synthesis of 2'-modified sugars or-carbocyclic sugar analogs, are described in Sproat, B.S. et al., Hue Acifl &1A (1991) Hl733-738; Cotten, M. et al., Nuc Add Rea (1991) 12:2629-2635; Hobbs, J. et al., Biochemistry (1973) 12:5138-5145; and Perbost, M. at al., Biochem Biophys Rea Comm (1989) 142:742-747 (carbocycliee).

Methods to Prepare the Invention Aptamers In general, the method for preparing the aptamers of the invention involves Incubating thrombin with a mixture of oligonucleotides under conditions wherein some but not all of the members of the oligonucleotide mixture form ccoplexes with the thrombin. The resulting complexes are then separated from the uncomplexed members of the oligonucleotide mixture and the complexed members which constitute an aptamer (at this stags the aptamer generally being a population of a multiplicity of oligonucleotide sequences) is recovered from the complex and amplified. The resulting aptamer (mixture) may then be substituted for the starting mixture is repeated iterations of this series of steps.

When satisfactory specificity is Obtained, the aptamer may be used as a obtained or may be sequenced and synthetic forms of the aptamer prepared. In thie most gonoralieed form of the method, the oligonucleotides used as members of the starting mixture may be single-a trended or double-stranded ONA or ANA, or modified fonts thereof. -ιβBowever, single-stranded DNA is preferred. The uee of DNA eliminates the need for conversion of RNA aptamere to DNA by reverse transcriptase prior to PCR amplification.

Furthermore, DNA is less susceptible to nuclease degradation than RNA.

The starting mixture of oligonucleotide may be of undetermined sequence or may preferably contain a randomised portion, generally including from about 3 to about 400 nucleotides, more preferably 10 to 100 nucleotides. The randomization may be complete, or there may be a preponderance of certain sequences in the mixture, or a preponderance of certain residues at particular positions. Although, as described hereinbelow, it is not essential, the randomized sequence is preferably flanked by primer sequences which permit the application of the polymerase chain reaction directly to the recovered oligonucleotide from the complex. The flanking sequences may also contain other convenient features, such as restriction sites which permit the cloning of the amplified eequence. These primer hybridisation regions generally contain 10 to 30, more preferably 15 to 25, aad most preferably IB to 20, bases of known sequence.

The oligonucleotides of the starting mixture may be conventional oligonucleotides, most preferably single-stranded DNA, or may be modified forms of these conventional oligomers as described hereinabove. For ί oligonucleotides containing conventional phoaphodieeter [ linkages or closely related forms thereof, etandard j oligonucleotide synthesis techniques may bs employed. I Such techniques are well known in the art, such methods being described, for example, in Froehler, B., et al., Nucleic Acide Research (1986) 11:5399-5467; Nucleic Acids Research (1988, 11:4831-4839; Nucleoeldas and Nucleotides (1987) 1:287-291; Froehler, B., Tet Lett (1986) 12)5575IE 920561 -175578. Oligonucleotides may also be synthesised using solution phase methods such as triester synthesis, known in the art. The nature of the mixture is determined by the manner of the conduct of synthesis. Randomisation can be achieved, if desired, by supplying mixtures of nucleotides for ths positions at which randomization is desired. Any proportion of nucleotides and any desired number of such nucleotides can be supplied at any particular step. Thus, any degree of randomisation may be employed. Some positions may be randomised by mixtures of only two or three bases rather than tha conventional four. Randomised positions may alternate with those which have been specified. It may be helpful if some portions of ths candidate randomized sequence are in fact known.

In one embodiment of the method of the invention, the starting mixture of oligonucleotides subjected to the Invention method will have a binding affinity for thrombin characterized by a kd of l μΜ or greater. Binding affinities of the original mixture for thrombin may range from about 100 μΜ to l μΜ but, of course, the smaller the value of the dissociation constant, the more initial affinity there is in the starting material for thrombin. This may or may not be advantageous as specificity may be sacrificed by starting the procedure with materials with high binding affinity.

By application of the method of the invention as described herein, improvements in ths binding affinity over one or several iterations of the above steps of at least a faetor of 50, preferably of a factor of 100, and more preferably of a factor of 200 may be achieved. As defined herein, a ratio of binding affinity reflects ths ratio of kds of the comparative complexes. Bven more preferred in the conduct of the method of the invention -Ιβia the achievement of an enhancement of an affinity of a factor of 500 or aore.

Thue, the method of the invention can be conducted to obtain the invention aptamers wherein the aptamers are characterized by consisting of singlestranded dna, hy having a binding affinity represented by a Kg of 10'9 or less, by having a specificity representing by a factor of at least 10 with respect to unrelated molecules, by having a binding region of less than 14 nucleotide residues or a total size of less than 15 nucleotide residues, or by binding to thrombin. The invention processes are also characterized by accommodating starting mixtures of oligonucleotides having a binding affinity for thrombin characterized by a kd of 1 μΜ or more by an enhancement of binding affinity of 50 or more, and by being conducted under physiological conditions. As used herein, physiological conditions means the salt concentration and ionic etrength in an aqueous solution which characterize fluids found in human metabolism commonly referred to as physiological buffer or physiological saline. Zn general, these are represented by an intracellular pH of and salt concentrations_and an extracellular pH of _and salt concentrations_. msLflf Modified WucleotldcB and Qilgonucleotldei Zn one embodiment of the invention method, the initial mixture of candidate oligonucleotides will include oligomers which contain at least one modified nucleotide residue or linking group.

If certain specific modifications are included in the amplification process as well, advantage can be taken of additional properties of any modified nucleotides, such as the presence of specific affinity agents in the purification of the desired materials. -ι»In order for the modified oligomer to yield useful results, the modification must result in a residua which is read in a known way hy the polymerising enzyma used in the amplification procedure. Zt is not necessary that the modified residue be incorporated into the oligomers in the amplification process, as long it is possible to discern from the nucleotide incorporated at the corresponding position the nature of the modification contained in the candidate, and provided only one round of complexation/amplifieation is needed. However, many of the modified residues of the invention are also susceptible to ensymatic incorporation into oligonucleotides by the commonly used polymerase enzymes and the resulting oligomers will then directly read on IS the nature of the candidate actually contained in the initial conplex. It should bs noted that if mors than one round of coeplexation is needed, the amplified sequence must include the modified residue, unless the entire pool is sequenced and resynthesized to Include the modified residue.

Certain modifications can bs made to the base residues in a oligonucleotide sequence without iopairing the function of polymerizing enzymes to recognise the modified bass in the template or to incorporate the modified residue. These modifications include alkylation of the 5-position of uridine, deoxyuridine, cytidine and deoxyoytidine; the N^-position of cytidine and deoxycytidine; the M*-position of adenine and deoxyadenine; the 7-poettion of deazaguanine, deasadeoxyguanlne, deazaadenine and deasadeoxyadenine.

As long as the nature of the recognition ls known, the modified bass may bs included in the oligomeric mixture! useful in the method of the invention.

The nature of the sugar moiety may also bs modified without affecting the capacity of tha aaqusnce -20to be usable as a specific template in the synthesis of new DNA or RNA.

The efficacy of the process of selection and amplification depends on the ability of the PCR reaction faithfully to reproduce the sequence actually complexed to thrombin. Thus, if the oligonucleotide contains modified forms of cytosine (C*), the PCR reaction must recognize this as a modified cytosine and yield an oligomer in the cloned and sequenced product which reflect this characterization, if the modified form of cytosine (C*) is included in the PCR reaction as dC*TP, the resulting mixture will contain C* at positions represented by this residue in the original member of the candidate mixture. (It is seen that the PCR reaction cannot distinguish between various locations of C* in the original candidate; all C residua locations will appear as C*.) Conversely, dCTP could be used in the PCR reaction and it would be understood that one or more of the positions now occupied by C was occupied in the original candidate mixture by C*( provided only one round of complexation/aaplification is needed. If the amplified mixture is used in a second round, this new mixture must contain the modification.

Of course, if the selected aptamer is sequenced and resynthesized, modified oligonucleotides and linking groups may arbitrarily by used in the synthesized form of the aptamer.

Inclusion of modified oligonucleotides in the methods and aptamers of the Invention provides a tool for expansion of the repertoire of candidates to include large numbers of additional oligonucleotide sequences. Such expansion of the candidate pool may be especially important as the demonetration of binding to proteins, for exaaple, in the prior art ie limited to those proteins known to have the capability to bind DNA. -21Modifications of the oligonucleotide may be necessary to include all desired sequences among those for which specific binding can be achieved.

Thus, one preferred method comprises incubating 5 thrombin with a mixture of oligonucleotides, wherein these oligonucleotides oontain at least one modified nucleotide reeidue or linkage, under conditions wherein complexation occurs with some but not all members of the mixture; separating the complexed from uncomplexed oligonucleotides, recovering and an^lifying ths complexed oligonucleotides and optionally determining ths sequence of the recovered nucleotides. In an additional preferred embodiment, amplification is also conducted in the presence of modified nucleotides.

A Subtraction Method for Aptamer Preparation It is often advantageous in enhancing the specificity of the aptamer obtained to remove members of the starting oligonucleotide mixture which bind to a second substance from which thrombin is to be distinguished. In such subtraction methods, at least two rounds of selection and amplification will be conducted. Xn a positive/negative selection approach, thrombin will be incubated with the starting mixture of oligonucleotides and, as usual, the complexes form separated from uncomplexed oligonucleotides. The complex oligonucleotides, which art now an aptsmsr, art recovered and amplified from the complex. The recovered aptamer is then mixed with the second undesired substance from which thrombin is to be distinguished under conditions wherein members of the aptamer population which bind to said second substance can be complexed. This complex is then separated from the remaining ollgonucleotidee of the aptamer. The resulting second aptamer population is then recovered and amplified. The second aptamer population -22ie highly specific for thrombin as compared to the second substance.

In an alternative approach, the negative selection step may be conducted first, thus mixing the original oligonucleotide mixture with the undeeired substance to eouplex away the members of the oligonucleotide mixture which bind to the second substance; the uncomplexed oligonucleotides are then recovered and amplified and incubated with thrombin under conditions wherein those members of the oligonucleotide mixture which bind thrombin are complexed. The reeulting complexes then removed from the uncomplexed oligonucleotides and the bound aptamer population is recovered and amplified as usual.

Modified Method Wherein Thrombln/Aptamer Complexes are Separated from Solid Support As set forth hereinabove, the original oligonucleotide mixture can be synthesized according to the desired contents of the mixture and can be separated by adding the oligonucleotide mixture to a column containing covalently attached thrombin (see, BI ling ton, A.D., et al., Nature (1990) 818-822, or to thrombin in eolution (see Blackwell et al., Science (1990) :1104-1110; Blackwell et al., Science (1990) 252:11491151; or to thrombin bound to a filter (see Tuerk, C., and Sold, L., fifiilOSl (1990) 212:505-510). Complexes between the aptamer and thrombin are separated from uncomplexed aptamere using any suitable technique, depending on the method used for complexation. For example, if columns are used, non-binding species are simply washed from the column using an appropriate buffer. Specifically bound material can then be eluted. if binding occurs ln solution, the complexes can be separated from the uncomplexed oligonucleotides -33using, for example, the mobility shift in electrophoresis technique (BM8A), described in Davis, R.L., at al., Cell (1990) £&i733. in thie method, aptamer-thrombin complexes are run on a gel and aptamers removed from the S region of the gel where thrombin runs. Unbound oligomers migrate outside these regions and are separated away. Finally, if complexee are formed on filters, unbound aptamers are eluted ueing standard techniques and the desired aptamer recovered from the filters.

Zn a preferred method, separation of the complexes involves detachment of thrombin-aptamer cooplexes from column matrices as follows. λ column or other support matrix having covalently or noncovalently coupled thrombin is synthesised. Any standard coupling reagent or procedure may be utilised, depending on the nature of the support. For example, covalent binding may include the formation of disulfide, ether, ester or amide linkages. The length of the linkers used may be varied by conventional means.

None ova lent linkages include antibody-antigen interactions, protein-sugar interactions, as between, for example, a lectin column and a naturally-occurring oligosaccharide unit on a peptide.

Lectin columns are particularly suited for selecting thrombin aptamers. Lectins art proteins or glycoproteins that can bind to complex carbohydrates or oligosaccharide units on glycoproteins, and are welldescribed in The Lectins (I.I. Liener et al., ed·., Academic Press 1986). Lectins are Isolated from a wide variety of natural sources, including peas, beans, lentils, pokeweed and snails. Concanavalln A is a particularly useful lectin.

Other linking chemistries are also available. For exanplt, disulfide-derivatlied biotin (Pioroo) may bo linked to thrombin by coupling through an amine or other -24functional group. Th· reeultlng thrombin-S-S-biotin complex could then be used in combination with avidinderivatized support. Oligonucleotide-thrombin complexes could then be recovered by disulfide bond cleavage.

Linking chemistries will be selected on the basis of (i) conditions or reagents necessary for maintaining ths structure or activity of thrombin.

The oligomer mixture is added to and incubated with the support to permit oligonucleotide-thrombin complexation. Complexes between the oligonucleotides and thrombin are separated from uncomplexed oligonucleotides by removing unbound oligomers from the support environment. For example, if columns are used, nonbinding specie· are simply washed from the column ueing an appropriate buffer.

Following removal of unbound oligomers, the thrombin ls uncoupled from the eupport. The uncoupling procedure depends on the nature of the coupling, as described above. Thrombin bound through disulfide linkages, for sxaaple, may be removed by adding a sulfhydryl reagent such as dithiothreitol or 3· mercaptoethanol. Thrombin bound to lectin supports may ba removed by adding a complementary monosaccharide (e.g., α-methyl-mannoside, N-acetyl glucosamine, glucose, N-acetyl galactossmine, galactose or other saccharides for concanavalin A). oligonucleotides specifically bound to thrombin can than bs recovered by etandard denaturation techniques such as phenol extraction.

The method of elution of thronbln30 oligonucleotide conplex from a support has superior unexpected properties when coopered with standard oligonucleotide elution techniques. This invention is not dependent on the mechanism by which these superior properties occur. However, without wishing to be limited by any one mechanism, the following explanation ls -25offered ae to how more efficient elution le obtained. Certain support effects result from the binding of oligonucleotides to the support, or the support in conjunction with oligonucleotide or thrombin. Removing oligonucleotide-thronbin complexes enables the recovery of oligonucleotides specific to thrombin only, while eliminating oligonucleotides binding to the support, or the support in conjunction with oligonucleotide or thrombin. At each cycle of selection, this method may give up to 1,000-fold enrichment for specifically binding species. Selection with thrombin remaining bound to support gives less enrichment per cycle, making it necessary to go through many more cycles in order to get a good aptamer population.

Aptamer Poole of Varying Length Aptamers can also be selected in the above methods using a pool of oligonucleotides that vary In length as the starting material. Thus, several pools of oligonucleotides having random sequences are synthesised that vary in length from e.g. 50 to 60 bases for each pool and containing the same flanking primer-binding sequences. Bqual molar amounts of each pool are mixed and the variable-length pool is then used to select for aptamers that bind to thrombin, as described above. This protocol selects for the optimal species for thrombin binding from the starting pool and does not limit aptamers to those of a given length.

Alternatively, several pools of mixed length aptamers can be used in parallel ia separate selections and then combined and further selected to obtain the optimal binders from the size range initially used. For exaaq>le, three pools, A, B and C, can be used. Pool A can consist of oligonucleotides having random sequences that vary in length from e.g. 30 to 40 baeeej pool B can -24have sequences varying in length from e.g. 40 to 50 bases; and pool C can have sequences varying in length from 50 to 40 bases, it is to be understood that the lengths described above are for illustrative purposes only. After selection to obtain binders from A, B, and C, all aptamers are mixed together. A number of rounds of selection are done as described above to obtain the best binders from the initial species selected in the 30» to 40-base range. Note that with thie technique, not all possible species in some of the pools are used for selection, if the number of sites available for binding are increased, i.e., if a column is used and the size of the column increased, more species can bs included for selection. Furthermore, this method allows for the selection of oligomers from the initial starting pool that are of optimal length for binding thrombin.

The oligonucleotides that bind to thrombin are separated from the rest of the mixture and recovered and amplified. Anplification may be conducted before or after separation from thrombin. The oligonucleotides ars conveniently amplified by PCR to give a pool of dna sequences. The PCS. method is well known in the art and described in, e.g., u.8. Patent Nos. 4,443,195 and 4,483,202 and 8aiki, R.K., et al., Sclencs (1988) 221:487-491, and Buropeen patent applications 84302298.4, 84302299.2 and 87300203.4, as well as Methods in gnsvmologv (1987) 151:335-350. If RNA is Initially used, the amplified DNA sequences are transcribed into RNA.

The recovered DNA or RNA, in the original single-stranded or duplex form, is then used in another round of selection and amplification. After three to six rounds of selectlon/amplification, oligomers that bind with an affinity in the mM to μΚ range can be obtained and affinities below the μϋ range are possible. PCR may also be performed in the presence of thrombin. 27Other methods of amplification may be employed Including standard cloning, ligase chain reaction, etc. (See e.g., Chu, et al., U.S. Patent No. 4,957,858). For example, to practice this invention using cloning, once the aptamer haa been identified, linkers may be attached to each side to facilitate cloning into standard vectors. Aptamers, either in single or double stranded form, may be cloned and recovered thereby providing an alternative amplification method.

Amplified sequences can be applied to sequencing gels after any round to determine the nature of ths aptamers being selected by thrombin. The entire process then may be repeated using the recovered and amplified duplex if sufficient resolution is not obtained.

Amplified sequences can be cloned and Individual oligonucleotides then sequenced. The entire process can then be repeated using the recovered and amplified oligomers as nssded. Once an aptamer that binds specifically to thrombin has been selected, it may be recovered as DNA or RNA in single-stranded or duplex form uelng conventional techniques.

Similarly, a selected aptamer may be sequenced and resynthesized using one or more modified bases, sugars and linkages using conventional techniques. The specifically binding oligonucleotides need to contain the sequence-conferring specificity, but may be extended with flanking regions and otherwiee derivatized.

Derlvatlzatlon Aptamers containing ths specific binding sequences discerned through the method of the invention can aleo be derivatized in various ways. For example, lf the aptamer is to be used for separation of thrombin, conventionally the oligonucleotide will be derivatized to -21a solid support to permit chromatographic separation. If the oligonucleotide ia to be used for attaching a detectable moiety to thrombin, the oligonucleotide will be derivatized to include a radionuclide, a fluorescent molecule, a chromophore or the like. If the oligonucleotide is to be used in specific binding assays, coupling to aolld support or detectable label, and the like are also desirable, if it is to be used in therapy, the oligonucleotide may be derivatized to include ligands which pennifc easier transit 9t gfllular barriers, toxic moieties which aid in the therapeutic effect, or enzymatic activities which perform desired functions at the thrombin site. The aptamer may aleo be included in a suitable expression system to provide for In situ generation of the desired sequence.

Consensus Sequences When a number of individual, distinct aptamer sequences for thrombin have been obtained and sequenced as described above, the sequences may be examined for consensus sequences. As used herein, consensus sequence refers to a nucleotide sequence or region (which may or may not be made up of contiguous nucleotides), which is found in on· or more regions of at least two aptamers, the presence of which may be correlated with aptamer-to-thrombin-binding or with aptamer structure.

A consensus sequence may be as short as three nucleotides long. It also may be made up of one or more noncontiguous sequences with nucleotide sequences or polymers of hundreds of bases long interspersed between the consensus sequences. Consensus sequences may be identified by sequence comparisons between individual aptamer species, which comparisons may be aided by computer programs and other tools for modeling secondary -29and tertiary structure from sequence information. Generally, the consensus sequence will contain at least about 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.

When a consensus sequence is identified, oligonucleotides that contain that sequence may be made by conventional synthetic or recombinant means. These aptamere, termed secondary aptamere, may also function as thrombin-specific aptamere of this Invention. A secondary aptamer may conserve the entire nucleotide sequence of an Isolated aptamer, or may contain one or more additions, deletions or substitutions in the nucleotide sequence, aa long as a consensus sequence is conserved. A mixture of secondary aptamere may also function as thrombin-specific aptsmers, wherein the mixture is a set of aptamere with a portion or portions of their nucleotide sequence being random or varying, and a conserved region which contains the consensus sequence. Additionally, secondary aptamere may be synthesised using one or more of the modified bases, sugars and linkages described herein using conventional techniques and those described herein.

Utility oC tht Aptaiwri The aptamere of the invention are useful ln diagnostic, research and therapeutic contexts. For therapeutic applications, the thrombin aptamers have la vivo and ex vivo clinical utilities, as indicated above. By way of example, the aptamers may be used ln the treatment or prevention of (i) restenosis or myolntimal thickening associated with angioplasty, (il) accelerated atherosclerosis after heart transplant operations, (Hi) vascular graft reooclusion associated with vascular shunt implants, (iv) clotting or thrombus formation at the site of indwelling arterial or venoue access lines, (v) -30thrombus formation associated with cardiopulmonary bypass surgery, (vi) thrombus formation associated with extracorporeal circuits that are used during various fix vivo procedures such as blood dialysis or apheresis, (vii) sepsis-related disseminated intravascular coagulation and (viii) coagulation in patients with known heparin allergy or heparin-induced thrombocytopenia.

For diagnostic applications, these aptamers are well suited for binding to biomolecules that are identical or similar between different species, where standard antibodies may be difficult to obtain. They are also useful in inhibition assays when the aptamers are chosen to inhibit the biological activity of thrombin. Antibodies are generally used to bind analytes that art detected or quantitated in various diagnostic assays. aptamers represent a class of molecules that may be used in place of antibodies for ia vitro or ia vivo diagnostic and purification purposes.

Aptamers that bind to thrombin may be used as ia vivo imaging or diagnostic reagents when suitably radiolabeled. lootopee such as l31I, ®Tc, 90Y, 15,1ln and l23X have been used to label various proteins or antibodies as is described in the literature (Cohn, K.H., St al, Arch. Surg. (1907) 122: 1245-1429; Baidoo, R.B., et al, Cancer Bee. (Suppl.) (1990) 2£s799e-803s; Beatty, J.D., et al, Cancer Res. (Suppl.) (1990) £&:040s-045j Sharkey, R.M., et al e^cer ae·. (1988) 42*32270-3275).

A preferred isotope is ®Tc which is utilised as described in the literature. Chemical modifications of oligonucleotides that are compatible with labeling protocols are also known in ths art and have been extensively described (Uhlmann, B., et al, Chemical Rev. (1990) 22:543-584; international publication Roe. wo 91/14696 and WO 91/13080). -31The thrombin aptamer· may alao be labelled by > linking a moiety that chelate· aa imaging agent euch a· 99eTc. In thi· embodiment, thrombin aptamer would be administered to a patient followed by administration of ths imaging agent, in vivo chelation of the imaging agent would occur, allowing subsequent imaging by conventional means.

Thrombin aptamers may also be labeled with contrast agents such as lanthanide or transition metal conplexea or nuclei euch as 19F, liN or ,aP to facilitate in vivo imaging of clots and similar formations. Imaging would be performed ueing magnetic resonance imaging techniquee known in the art.

One consideration in generating radiolabeled antibodies is that the labeling procedure must not destroy its antigen-binding properties. This usually requires an optimized protocol to be generated for each isotope and antibody. Because the aptamers of ths invention are tolerant of harsh chemical conditions, including conditions under which they ars synthesized, facile radiolabeling of thrombin aptamers can bs conducted without regard to loss of aptamer structure.

Only ths chemical integrity of the aptamer molecule must be preserved. The aptamers of ths invention can be denatured without loss of their ospacity to bind thrombin ones placed under physiological conditions. Antibodies cannot be reversibly denatured in this manner.

Another consideration relevant to the use of monoclonal antibodies (MAbs) for in vivo imaging is their antigenicity. MAbs ire usually derived from mouse hybridomas and as such are foreign proteins. When used in humans they elicit immune responses that limits their use in individual patients to one or two exposures. Once immunised, anti-NAb antibodies generated by an immunized -32individual leads to rapid clearance of the MAb. Thia consideration io aloo rolavant to humanized Mhhe rhat. contain both mouse and human protein sequences.

In addition to chemical stability, the aptamers 5 described herein have a short half-life, a property that can permit rapid ia vivo imaging after adminletration of labeled compound. Ihe thrombin aptamers can also be advantageously used to avoid anaphylactic reactions such as those associated with imaging procedures that use conventional ionic or nonionie contrast agents. The aptamers also have a low molecular weight compared to Abs, which can facilitate their penetration of a target structure, such as a clot, for imaging purposes.

Radiolabeled thrombin aptamers can be used to image arteries or veins according to various clinical indications. For exaaple, such aptamers can be used after angioplasty to image clots, including deep vein clots, CMS thromboses, pulmonary emboli, brain thrombosea and the like.

The aptamers of the invention are therefore particularly ueeful ae diagnostic reagents to detect ehe presence or absence of thrombin. Xa yitlfl diagnostic tests are conducted by contacting a sample with the specifically binding oligonucleotide to obtain a complex which is then detected by conventional means. For example, the aptamers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support to which the thrombin has been bound through a specific or nonspecific binding means detected. Alternatively, the specifically binding oligonucleotides may be used to effect initial complexation to the support. Means for conducting assays using such oligomers as specific binding partners will track those for standard specific binding partner based assays. 3)It may ba commented that the mechanism by which the specifically binding oligomers of the invention interfere with or Inhibit the activity of thrombin ie not always established, and is not a part of the invention.

The oligomere of the Invention are characterized by their ability to bind thrombin regardless of the mechanisms of binding or the mechanism of the effect thereof.

For use in research or manufacturing, the specifically binding oligonucleotides of ths invention are especially helpful in effecting the isolation and purification of substances to which they bind. For this application, typically, the aptamer containing the specific binding sequences is conjugated to a solid support and used as an affinity ligand in chromatographic separation of thrombin.

Xn therapeutic applications, the aptamers of the invention can be formulated for a variety of modes of administration, including systemic and topical or localised administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences. Mack Publishing Co., Faston, PA, latest edition. Xn general, the dosage required for therapeutic efficacy will range from about 0.1 pg to 20 mg aptamer/kg body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.

For systemic administration, injection is preferred, including intramuscular, Intravenous, intraperitoneal, and subcutaneous. For injection, the aptamers of the invention are formulated in liquid solutions, preferably in physiologically conipatible buffers such as Rank's solution or Ringer's solution. Xn addition, the aptamers may be formulated in solid form and -34redissolved or suspended immediately prior to use. Lyophilized forms are also included.

Systemic administration can also be by transmucosal or transdezmal means, or the oligomers can be administered orally. Additional formulations which are suitable for other modes of administration include suppositories, Intranasal and other aerosols. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are ueed in the formulation. Such penetrants are generally known In the art, and include, for example, for transmucosal administration bile ealte and fuaidic acid derivatives.

In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays, for example, or using suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams, ae is generally known in the art.

The aptamers may also bs employed in expression systems, which are administered according to techniques applicable, for instance, in applying gene therapy.

The following examples are meant to illuatrate, but not to limit the invention. wimple 1 flelection of Antamers that Bind to Thrombin 30 a. Synthaaia of QliggauclaatldaPtx?l DNA oligonucleotides containing a randomized sequence region were eyntheeised ueing standard solid phase techniques and phosphoramidite chemistry (Oligonucleotide Synthesis. Oait, M.J., ed. (IRL Press), -351964; Cocuzza, λ., Tetrahedron Latter·. (1969) 12:62876291). λ 1 μΜ small-scale synthesis yielded 60 nmole of HPLC-purified single-stranded randomized DNA. Bach strand consisted of specific 18-mer sequences at both the * and 3' ends of the strand and a random 60-mer sequence ln the center of the oligomer to generate a pool of 96-mere with the following sequence (N - G, A, T or C): ' HO-CGTACGGTCGACGCTAGCNeoCACGTGGAGCTCGGATCC-OH 3' DNA 16-mere with the following sequences were used as primers for PCR amplification of oligonucleotide sequences recovered from selection columns. The 5* primer sequence was 5' KO-CGTACGGTCGAOOCTAGC-OK 3' and IS the 3' primer sequence was 5' biotin-0GGATCCGAOCTCCACGTG-OH 3'. The biotin residue was linked to the 5* end of the 3' primer using commercially available biotin phosphoramidite (New England Nuclear, Cat. No. NBP-707). The biotin phosphoramidite is incorporated into the strand during solid phase DNA synthesis using standard synthesis conditions.

In another, similar experiment, a pool of 98-mere with the following eequence was synthesised: ' HO-AGAATACTCAAGCTTGCCG-N60-KCCTGAATTCGCCCTATAO-OH 3'.

DNA 19 *mers with the following sequences can also be used as primers for PCR amplification of oligonucleotides recovered from selection columns. The 3' primer sequence is S' biotin-O-CfTATAGGGCGAATTCAGGT-OH 3' and the 5’ primer sequence is IE 92056T •36* * HO-AGAATACTCAAGCITGCCG-OH 3*.

It will be noted that in all cases, the duplex form of the primer binding sites contain restriction enzyme sites.

B. Isolation of Thrombin Aptamers Using Thrombin Immobilized on a Lectin Column λ pool of aptamer dna 96 bases in length wae synthesized as described in Bxample 1-A, and than PCRamplified to construct ths initial pool, λ small amount of the enzymatically-synthesized DNA was further amplified in the presence of e«32P-dNTFe to generate labeled aptamer to permit quantitation from column fractions.

A thrombin column was prepared by washing 1 ml {58 nmols) agarose-bound concanavalin A (Con-A) (Vector Laboratories, cat. no. AL-1003) with 20 mM Tris*acetate buffer (pH 7.4) containing 1 mN MgCl2, 1 mN CaClj, 5 mN XC1 and 140 mM NaCl (the selection buffer) (4 x 10 ml). 1 ml of settled support was then incubated overnight at 4C in 10 ml selection buffer containing 225 /ig (6.25 nmols) thrombin (Sigma, Cat. no. T-6759). After shaking overnight to permit thrombin binding to the Con-λ beads, ths mixture was briefly centrifuged and ths supernatant removed. The beads were resuspended in fresh selection buffer and transferred to a column which was then washed with selection buffer (5 χ 1 ml). A column containing 1 nt of settled beads had a void volume of approximately 300 pL. A control Con-A column was prepared by adding 1 mt of settled support to a column followed by 5 washes of l ml of selection buffer.

Prior to application of the aptamer DNA pool to Con-λ columns, the DNA was heated in selection buffer at 95C for 3 minutes and then cooled on ice for 10 minutes. ie szoser -37The pool, consisting of 100 pmol· DNA in 0.5 mJ selection buffer, wae then pre-run on the control Con-A column at room temperature to remove speciee that bound to the control support. Three additional 0.5 mt aliquots of selection buffer were added and column fractions 2, 3 and 4 (0.5 nt each) were pooled and then reapplied to the column twice. The DNA in 1.5 nt selection buffer was then recovered. Approximately It of total input cpm were retained on the column.

The recovered DNA was then applied to a Con-Athrombin column ae a 0.5 mJ aliquot followed by a 1.0 mJ aliquot. Flow-through was retained and reapplied to the column twice. DNA added to the column on the final application was left on the column for 1 hour at room temperature. The column was then eluted with 0.5 mJ aliquots of sslsction buffer. 0.5 mJ fractions were collected and radioactivity was determined in each fraction. Radioactivity in eluted fractions 7 through 12 wore low and relatively constant. After recovery of fraction 12, the column was washed with 0.5 mJ aliquots of 0.1 M a-methyl-mannoeide (Sigma Cat. no. M-6882) in selection buffer to elute the bound thrombin along with thrombin*bound aptamers. Fractions 14 and 15 showed a significant peak of thrombin enzyme activity, as determined spectrophotometrically by conversion of a chromogenic substrate (Xabi Diagnostics, Cat. ao. 8-2238). 0.01% of the input DNA eluted in these two fractions.

Aptamer dna (Round 1 DNA) was recovered from the thrombin by phenol extraction (2 x 0.5 mJ). The aqueous phase volume was reduced to about 250 μΐ by nbutanol extraction. Aptamer DNA was precipitated on dry ice using 3 volumes of ethanol and 20 pg of glycogen as a carrier. The DNA was pelleted, washed once in 70% ethanol and then dried. -38C. Amplification of Selected Thrombin Aptamera Round l dna from Example 1-B was reeuepended in 100 μί of HjO and amplified by PCR. A 200 μί PCR 5 reaction consisted of the following: 100 μί template 96mer DNA (approximately 0.01 pinoles); 20 μΐ 10X buffer (100 nN Tria-Cl (pH 8.3), 500 mM XCl, 20 mM MgCla)j 32 μΐ dNTP'a (5 mM cone total, 1.25 mM each dATP, dCTP, dOTP, and dTTP) i 20 μί primer 1 (biotinylated 18*mer, 50 μΜ); μΐ primer 2 (18-mer, 50 μΜ); 6μ1 α-^^P-dNTP's (approximately 60 pCi); and 2 μΐ Tag X Polymerase (10 units). The reaction was covered with 2 drops NUJOL mineral oil. A control reaction was also performed without template aptamer.

Initial denaturation wae at 94 *C for 3 minutes, but subsequent denaturation after each elongation reaction lasted 1 minute. Primer annealing occurred at 60*C for 1 minute, and elongation of primed DNA etrande using the Taq polymerase ran at 72”C for 2 minutes, with -second extensions added at each additional cycle. The final elongation reaction to completely fill in all etrande ran for 10 minutes at 72*C, and the reaction was then held at 4*C. rounds of Taq polymerase elongation were carried out in order to amplify the selected aptamer DNA. After the reactions were completed, the aqueous layer wae retrieved and any residual NUJOL oil wae removed by abutanol extraction, reducing the volume to 100 μί. A sample may be removed from each of the aptamer and control reaction for quantitation and analytical PAQB.

The amplified aptamer pool (100 μί) wae run over a Nick column (0-50 Sephadex, washed with 3 mL Tl buffer (10 mM TrirHCl (pH 7.6), 0.1 mM BDTA)) to remove unincorporated NTP's, primers, and salt. 400 μϊ» of Tl buffer was then added to the column and the DNA pool was eluted from the -j»column with an additional 400 pL using TB buffer. (A sample may be removed from the eluent for quantitation and analytical PAOB.) The eluent (400 pL) waa loaded on an avidin agarose column (Vector Laboratories, Cat. No.

A-2010) (500 pL settled support, washed with 3 x 1 nl> Tris/NaCl buffer (0.1 M Trio, 0.1 M NaCl, pH 7.5)). Approximately 90¾ of the loaded radioactivity remained on the column. The column was washed with Tris/NaCl buffer (4 x 400 pi) and then the nonbiotinylated strand was eluted with 0.15 N NaOH (3 x 300 pL fractions). Mors than 45¾ of ths radioactivity on the column eluted in these three fractions. These fractions (900 pi) were combined and neutralised with approximately 3.5 pi of glacial acetic acid. The neutralised fractions were reduced to 250 pi by speed vacuum or butanol extraction and the nucleic acids wars precipitated with BtOH. The resultant pellet was dissolved in 102 pi selection buffer. A 2 pi sample was removed for quantitation and analytical PAOB. The resulting amplified Round 1 Pool was applied to a new Con-A-thrombin column as in Bxample 1-B to obtain Round 2 aptamers.

D. Characterisation of Round 1 Through Round 5 J In ao Aptamers Obtained from Selection on Lectin Columns Five rounds of thrombin aptsmsr selection and amplification wars carried out using Con-A-throcnbin columns as in Examples 1-B and 1-C. As shown in Table 1, the combined fractions 14 and 15 contained a maximum of about 10¾ of input DNA using the described conditions. lh 92056T -40Table l Round % DNA eluted by t α-methyl-mannoeide 8 DNA bound to control support 1 0.01 0.7 2 0.055 1.9 3 5.80 2.3 4 10.25 1.1 5 9.70 1.0 * 0.1 H «-methyl-mannoeide in selection buffer was added as fraction 13 in each elution, and fractions 14 and 15 were retained and the DNA amplified. Due to slow leeching of thrombin from the column, dna bound to 15 thrombin could also be seen in earlier fractions in rounds 3-5.

After amplification, round 5 aptamer DNA was analysed for specificity in a filter binding assay. Xn 30 this assay, nitrocellulose filters (1 cm diameter) prebound with salmon sperm DNA were used to bind either: (1) An uneelected 96>mer oligonucleotide dna pool, (2) unselected DNA with thrombin (60 pmol·), (3) Round 5 aptamer DNA and thrombin (60 pools), (4) Round 5 aptamer 35 DNA alone, or (5) Round 5 aptamer dna and ovalbumin (60 pmole). Xn each case 3.5 pmole of DNA was used and the incubation was in 200 μϊ» selection buffer at room temperature for 1 hour. The filter· were then washed 3 times with 3.0 nt of selection buffer and radioactivity 30 was counted to determine the amount of DNA that was retained as a thrombin complex. The results are shown in Table 2.

IE 92056T DNA * DNA Bound to Filter Table 2 Unselected 96-raer 0.08 Uhselected 96-mer + thrombin 0.06 Round 5 aptamer ♦ thrombin 20.42 Round 5 aptamer 0.07 Round 5 aptamer * ovalbumin 0.05 Uhselected DNA did not show significant binding to the thrombin while selected aptamer DNA bound to thrombin. Binding resulte show specific thrombin binding with no detectable ovalbumin binding.

Round 5 aptamer DNA was then amplified using the following 3' primer sequence: ' HO-TAATACGACTCACTATAOOOATCCQAOCTCCACGTO-OH 3' and the 5' 18-mer primer sequence shown in Bxample 1-A. The 36-mer primer was used to generate Internal BamHl restriction sits· to aid ia cloning. Tho amplified Round 5 aptamer DNA was then cloned into pGBN 32 (Promega). 32 25 of the resulting clonss were then amplified directly using the following 5* primer sequence: ' HO-CTCkUkOGTOQAOQCTAOC-OK 3' 30 and the 3' biotinylated 18-mer primer aequenee shown in Bxample l-A, and then sequenced.

Filter binding assays using aptamer DNA from 14 of the clones were used to determine the dissociation constants (Kp) for thrombin as follows: Thrombin ,- concentrations between 10 iM and 1 nM were incubated at -42room temperature in selection buffer for 5 minutes in ths presence of 0.08 pmole of radiolabeled 96-mer derived from cloned Round 5 aptamer DNA. After Incubation, the thrombin and aptamer mixture was applied to nitrocellulose filters (0.2 micron, 2.4 cm diameter) that were pretreated with salmon sperm DNA (1 mg/mJ DNA in selection buffer) and waehed twice with 1 mJ selection buffer. After application of thrombin mixture, the filters were waehed three times with 1 mJ selection buffer The radioactivity retained on the filters was then determined. Kg values for the individual donee ranged from 50 to >2000 nM.

The DNA sequence of the 60-nudeotide randomlygenerated region from 32 clones was determined in order to examine both the heterogeneity of the selected population and to identify homologous sequences.

Sequence analysis showed each of the 32 clones to be distinct. However, striking sequence conservation was found. The hexamer 5' QGTTGG 3' was found at a variable location within the random sequence in 31 of 32 clones, and five of the six nucleotides are strictly conserved In all 32. Additionally, in 28 of the 32 clones a second hexamer 5' OGNTGG 3*, where N ie usually T aad never C, is observed within 2-5 nucleotides from ths first hexamer. Thus, 28 clones contain the consensus sequence 5' GGNTGO (N) aGGNTGG 3* where s is an Integer from 2 to 5. The remaining 4 clones contain a close variant sequence* (a sequence differing by only a single base). A compilation of the homologous sequences ars shown in Figure l. It should be noted that DNA sequencing of several clones from the unselected DNA population or from a population of aptamers selected for binding to a different target revealed ao homology to ths thrombin* selected aptamers. From these data we conclude that this consensus sequence contains a sequence which is -43responeible either wholly or in pert, for conferring thrombin affinity to the aptamere.

Clotting time for the thrombin-catalysed conversion of fibrinogen (2.0 ng/mL in selection buffer) S to fibrin at 37*C was measured using a precision coagulation timer apparatus (Becton-Dickinson, Cat. nos. 84015, 44019, ¢4020). Thrombin (10 nM) incubated with fibrinogen alone clotted in 40 sec, thrombin incubated with fibrinogen and Pl nuclease (Boehringer-Mannheim, Indianapolis, IN) clotted in 39 sec, thrombin Incubated with fibrinogen and aptamer cions *5 (200 nM) clotted in 115 sec, and thrombin incubated with fibrinogen, clone *5 (200 nM) and Pl nuclease clotted in 40 sec. All incubations were carried out at 37*C using reagents prewarmed to 37*C. Aptamer DNA or, when present, Pl nuclease, was added to the fibrinogen aolution prior to addition of thrombin. These results demonstrated that (1) thrombin activity was inhibited specifically by Intact aptamer DNA and (ii) that inhibitory activity by aptamer did not require a period of prebinding with thrombin prior to mixing with the fibrinogen substrate.

Inhibition of thrombin activity was studied using a consensus-related sequence 7-mer, 5' 9GTTGGG 3', or a control 7-mer with the same base composition but different sequence (S' GGGGGTT 3'). Clotting times were measured using ths timer apparatus as above. Ths thrombin clotting time in this experiment was 24 sec using thrombin alone (10 nM), 25 sec with thrombin and the control sequence at 20 pM and 38 sec with thrombin plus the consensus sequence at 20 pM, indicating specificity for thrombin inhibition at the level of the 7-mer.

The inhibitory aptamers ware active at physiological temperature under physiologic ion conditions and were able to bind to thrombin in the -44preeenc· of th· fibrinogen substrata, a key requirement for therapeutic efficacy.

Bxample ..¾ Modified Thrombin Aptamers Modified forms of the single-stranded, thrombin consensus sequence-containing deoxynucleotide 15-mer described in Bxample 2, 5' GQTTGGT3TGGTTGG 3«, and a closely related 17-mer, were synthesised using conventional techniques. These aptamers for ths most part contain the identical nucleotide sequences, bases, sugars and phosphodiester linkages as conventional nucleic acids, but substitute one or more modified linking groups (thloate or MBA), or modified bases (uracil or 5-(1-pentynyl-2'-deoxy)uracil). The aptamers containing 5-(1-pentynyl)-2'-deoxyuridine were generated by replacing thymidine in the parent aptamere. Thrombin aptamers containing 5-(1-pentynyl)-2'-deoxyuridine were also obtained by selection as described in examples 8 and 9 below.

Independent verification of the x4 for the nonmodified 15-mer was made by determining the extent of thrombin inhibition with varying dna concentration. The data revealed 504 inhibition of thrombin activity at approximately the same concentration as ths derived x£, strongly suggesting that each bound thrombin was largely, if not completely, inhibited, and that binding occurred with a lti stoichiometry.

IE 92056T -45- Table 3 Compound X£ (nM) GGTTGGTGTGGTTQQ 20 οστταβτοταατταο·α·τ 35 ggttggtotogtt’g’o 40 oVtYgVtVtVoYtVo 280 GGTTGG (dU) G (dU) GGTTGG 15 GG (dV) TGGTGTGG (dU) TGG 80 GGTTOGTGTGGTD'GG 20 * indicates a thioate (i.e., P(O)fi) linkage * indicates a MBA linkage U* indicates 5-(l-pentynyl)uracil Example 3 Incorporation of 5-(l-pentynyl)-2'-deoxvurldlne Into Aptamer Candidate DNA 5- (l-pentynyl) -2' -deoxyuridine wae synthesized and converted to the triphosphate as described in Otvos, L., et al., Nucleic Acids Res (1987) 1763-1777. The pentynyl compound was obtained by reacting 5-iodo-2'deoxyuridine with 1-pentyne in the presence of palladium catalyst. 5-(1-pentynyl) ·2 * · deoxyuridine triphosphate 25 was then used as a replacement for thymidine triphosphate in the standard PCR reaction.

A pool of 96-met single-stranded DNA was synthesised, each strand consisting of specific 18-mer PCR primer sequences at both the S' and 3' ends and a 30 random 60-mer sequence in the center of the oligomer.

Details of synthesis of the pool of single-stranded DNA is disclosed ln Example i above. PCR conditions were the same ae those described above, with the following changes. dATP, dGTP and dCTP were all used at a -46concentration of 200 μΜ. The optimal concentration for synthesis of full-length 96-mer DNA via PCR using 5-(1pentynyl)-2' -deoxyuridine was 800 μΜ. Generation of PCRamplified fragments demonstrated that the Taq polymerase both read and incorporated the base as a thymidine analog. Thus, the analog acted as both substrate and template for the polymerase. Amplification was detected by the presence of a 96-mer band on an BtBr-etained polyacrylamide gel.

Banpla 4 Incorporation of Other Base Analogs Into Candidate Aptamer DNA ithyl, propyl and butyl derivatives at the IS 5-position of uridine, deoxyuridine, and at the N*-position of cytidine and deoxycytidine are synthesized using methods described above. Bach compound is converted to the triphosphate form and tested in the PCX assay described in Bxample l using an appropriate mixture of three normal deoxytriphosphates or ribotriphosphates along with a single modified base analog.

This procedure may also be performed with N6-position alkylated analogs of adenine and deoxyadenine, and the 7-position alkylated analogs of deazaguanine, deazadeoxyguanine, deaiaadenine and deazadeoxyadenine, synthesized using methods described in the specification. in Aptamer Containlnq_Substltuts InternuclftQtide Linkage· Modified forms of the 15-mer thrombin aptamer, ' ggttqgtgtggttgg 3' containing one or two formacetal internucleotide linkages (O-CHj-O) in place of the phosphodiester linkage (O-PO(O)-O) were synthesised and ΙΕ 92056Γ -47assayed for thrombin inhibition ae described above. The H-phosphonate dimer synthon wae synthesized ae described in Matteucci, M.D., Tet, Lett.. (1990) 11:2385-2387. The fozmacetal dimer, s' T-O-CH^-O-T 3», was then used in solid phase synthesis of aptamer DNA. Control unmodified aptamer dna was used as a positive control.

The results that were obtained are shown in Table 4.

Table < Coopound dot time (sec) 100 nM 20 nM 0 : OOT TOGTOTOOTTOO 105 51 • a OOTTOOTOTOOT TOO 117 48 • a OOT TOOTOTOOT TOO 84 60 » «1 OOTTOOTOTOOTTOO 125 49 • e NO DNA CONTROL a a a a 25 indicates a formacetal linkage Thrombi, Aptamer ContiinlM Abaiic Nucleotide Residues Modified forme of the 15-mer thrombin aptamer, 25 5* OOTTOOTOTOOTTOO 3' containing one abaslc residue at each position in the aptamer were synthesised and assayed for thrombin inhibition ae described above. The abaslc residue, l,4-anhydro-2-deoxy-D-ribitol was prepared as deecribed in Britja, R., et al, ffiifllftnnirti· and 30 Nucleotides (1987) £:803-814. The Ν,Ν-dileopropylamino cyanoethylphosphoramldite synthon was prepared by standard methods as described in Caruthers, M.K. Accounts ~ i. Rea. (1991) ££:278-284, and the derivatised COP support was prepared by the procedures- deecribed in 35 Dahma, M.J., et al, Nucleic Adds Res, (1990) l£:3813. -48Ths «basic residua was singly substituted into tach of the 15 positions of ths 15-mer. Control unmodified aptamer dna was used as a positive control. The results that were obtained are shown in Table S.

Table S Compound clot time (sec) 100 nM 0 nM OGTTGOTGTGGTTGX 27 GOTTGGTGTGGTTXG 27 GOTTOQTQTQQTXOG 27 GGTTQGTGTGCXTQO 56 GGTTGGTGTGXTTGO 27 15 QGTTGGTGTXGTTGG 29 oottggtgxggtigg 43 οοτταοτχταοτταα 51 GGTTGGXQTQQTTOQ 161 GGTTGXTGTGGTTGO 27 20 ggttxgtgtggttqg 27 GGTXGGTGTGGTTGO 27 ggxtggtgtgottgo 62 GXTTGGTGTGGTTGG 27 xgttggtgtggtigg 28 25 oottoototoottoo 136 NO DNA CONTROL - 26 x - Indicates an «basic residue fflttlBPlt 7 Thrombin Aptaaeri-CoBtalnliffl (l-Propynyl) -2«-deoxyuridine Nucleotide Residues Modification of the 18-mer thrombin aptamer, 5 αοτταοτοταοττοο 3* to contain 5-(1-propynyl) -2'35 deoxyuridine nucleotide analogs at the Indicated it 920561 -49positions in th· aptamer wae effected hy the synthesis of these aptamers. They were assayed for thrombin inhibition as described above. The aptamer and the K-phosphonate were prepared as described in DeClercq, B., et al, J. Med.Chenin (1983) 2£:661-666; Froehler, B.C., et al, Nucleosides and Nucleotides (1987) £:287-291) and Froehler, B.C., et al, Tet. Lett. (1986) 22:469. This analog residue was substituted at the indicated positions and the aptamer assayed for Inhibition of thrombin. The results that were obtained are shown in Table 6.

Conpound clot time (sec) 100 nM 0 &M OOTTGOTSTOGT2QO οοττοοτοταοζτοα ogttgotozgwttqo οατταοζοτοοττοο οοτζαοτοταοτταο αοζταοτοτοοτταο QOTTOGTOTGQTTOO NO DNA CONTROL 147 129 120 118 187 138 125 Z - indicates a 5-propynyl-2'-deoxyuridine residue Incorporation of 5-(1-psntvnvl)-2'-deoxvurldlne -(1-pentynyl)-2'-dsoxyuridine was synthesised and converted to ths triphosphate as described in Otvos, L., st al., Nucleic Acids Res (1987) 1763-1777. The psntynyl compound was obtained by reacting 5-iodo-2'· deoxyuridins with l-pentyne in the presence of a palladium catalyst. 5-(1-pentynyl)-2’-deoxyuridine IE 920561 -50* triphosphate wae then ueed as a replacement for thymidine triphosphate in the standard PCR reaction. λ pool of 60-mer single*stranded DNA waa synthesized, each strand consisting of specific 18-mer PCR primer sequences at both the 5' and 3* enda and a random ϊυ-mer sequence in che center of the uliyutuvx. Details of synthesis of the pool of single-stranded dna is disclosed in Bxample 1.

Because of the poor substrate activity of 10 pentynyl dUTP when used with TAQ polymerase, VBNT™ thermostable polymerase, (New Bngland Biolabs, Cat. No. 254) was employed. Amplification was performed as per the manufacturers instructions. Pentynyl dUTP was Included in the reaction as a substitute for dTTP. The single-stranded 60-mer was isolated by a modification of standard procedurea. The 200 pL PCR amplification reaction was divided into two saaplee which were applied to two NICK* columns equilibrated (5 mL) as described.

The eluent was collected, pooled and applied to avidin* agarose as described. This column was washed with buffer followed by elution of single*etranded 60-mer dna with 0.15 N NaOH, pooled and neutralized with glacial acetic acid. Single-etranded 60-mer DNA was desalted on a MAPS column equilibrated in 20 mM Tris OAc (pH 7.4). 10X selection buffer ealte were added to the sasiple, heated to 95°C for 3 minutes, and transferred to wet ice for 10 minutes.

Bonrola 9 isolation of Thrombin Apt-emera Paine DNA Containing 5* (l-Pentvnvl) -2* -deoxvuridlne The pool of aptamer DNA 60 bases in length was used essentially as described ia Example 8. The aptamer pool aequenee was -51IE 92056T ' TAfiAATACTCAAGCrrCGAOG-NjQ-AOTTTGQATCCCCOGGTAC 3’, while the 5* primer sequence wae 5'TAGAATACTCAAOCTTCGACG 3' and the 3' biotin-linked primer was ' GTACCCGGGGATCCAAACT 3».

Thrombin immobilized on a Con-A lectin column served as the target as described.

After five rounds of selection, aptamer DNA was recovered and amplified using thymidine triphosphate (dTTP) in place of 5-(1-pentynyl) -2’ -deoxyuridine in order to facilitate subsequent cloning and replication of aptamer DNA in £«. aali. At this stage, the presence of a thymidine nucleotide at a given location in an aptamer corresponded to ths location of a 5-(1-pentynyl)-2*15 deoxyuridine nucleotide in each original round five aptamer. Thus, dTTP served to mark the location of 5(l-pentynyl)-2'-deoxyuridine residue! in the original selected DNA pools.

The round five amplified dna containing dTTP was digested with BamHI and Hindi!! and cloned into the corresponding sites of pGSM 3Z (Promega Biotech) and transformed into I. coll. DNA from 21 clones was analysed by dideoxy sequencing. Three of the clones contained aptamer sequences that were identical. Only one of the 21 donee contained a sequence that closely resembled the original 5' GGTTGG 3' binding motif obtained uelng thymine in the selection protocol.

One of these two clones (#17) and the original unselected pool was analysed for thrombin binding by nitrocellulose filter assay describee above using una labeled with 32P to pezmit analysis of thrombin binding characteristics. The labeled DNA was synthesized by PCX and contained 5-(1-pentynyl)-2’-deoxyuridine in order to retain the original selected dna structures. Ths dna was incubated with thrombin at various concentrations between -5210 nM and 10 μΜ to obtain tha Kg values for thrombin binding. The Kg of the uneelected pool was >10 μΜ while the Xg of clone 17 wae 300 nM.

Radiolabeled clone 17 DNA was synthesized using thymidine in place of 5-(l-pentynyl)-2' -deoxyuridine and the resulting DNA had a Kg of >10 μΜ, demonstrating that the 5-(l-pentynyl) -2*-deoxyuracil heterocycle could not be replaced by thymine in the selected aptamer without loss of binding affinity.

Representative sequences that were obtained are as follows.

' TAGTATOTATTATGTGTAG 3' ' ATAGAGTATATATGCTGTCT 3' ' GTATATAflTATAGTATTGOC 3* ' AGOATATATGATATGATTCGG 3’ ' TACTATCATGTATATTACCC 3' ' CATTAAACOCGAOCTTPJTO 3* ' CTCCCATAATGCCCTAGCCG 3' ' GACGCACCGTACCCCOT 3' ' CACCAAACGCATTGCATTCC 3* * GTACATTCAGGCTGCCTOCC 3' ' TACCATCCCQTGGACGTAAC 3' ' GACTAAACGCATTGTGCCCC 3’ ’ AACOAAGGGCACGCCGGCTO 3' ’ ACGGATGGTCTGGCTGGACA 3» laolitioa.of TtarcriaU-Aptanftri tiling DNA-Contalning S-Mathyl-a*-dsoacYflytldlas -methyl-2’-deoxycytidine triphosphate was obtained commercially (Pharmacia, Cat. No. 27-4225-01) and used to synthesise dna containing random sequences 60 bases in length flanked by primers 19 bases in length.

The pool of aptamer DNA 91 bases in length was used -53sssentially a· described ia example l. Thrombin immobilized on a Con-λ lectin column served as the target as described.

Briefly, a 200 μΣ* PCR reaction wae set up 5 using: 10 mM Tris-HCl, pH 8.3 at 35° C, 1.5 mM MgClj, 50 mM NaCl and 200 μΜ of each of dATP, dGTP, dTTP and 5methyl-2* - deoxy cyt idine triphosphate. 20 μΟί each of e32P-dATP and dGTP were added to label the DNA. 1 nmole of 5' and 3' primer were added followed by addition of 0.2 pmole of 98-mer template pool DNA. Amplification was initiated by addition of 2 μΧ< (10 U) of Taq polymerase followed by sealing of the reaction with a mineral oil overlay. About 16 cycles of aaplification wars performed followed by a 10 minute final extension to consists all duplex synthesis.

Amplified DNA was recovered (100 μί aqueous phase), n-butanol extracted (650 μΐι) and applied to a Nick column prewashed with 5 mL of buffer containing 100 mM Tris-HCl pH 7.5 and 100 mM NaCl. Bluted DNA was applied to a 0.5 mL avidin-agarose column prewashed in the seme buffer and washed until DNA loss from the column was < 1000 cpm. Single etranded DNA was eluted from the avidin column by washing with 0.15 N Nacl and the eluate was neutralized to pH 7.0 using glacial acetic acid. The 98-mer DNA was exchanged into selection buffer on a second Nick column and, after heat denaturation for 3 min at 95® c followed by cooling on ice for 10 min, used in aptamer selection on thrombin lectin columns. 1 mL thrombin columns were equilibrated in selection buffer prior to addition of single-etranded DNA. The singleetranded DMA vae recirculated for three complete passes. Upon completion of the third pass the peak radioactive element was then applied to a 1 mL ConA/thrombin column (charged with 3 nmoles of thrombin). Radioactive single35 stranded 98-mer was applied three times to this matrix. -54At the third application, the column was stoppered and allowed to stand for 1 hr. The column wae then waehed with selection buffer and 0.5 mL aliquot fractions fiollaor.fld. A tntfll wR«h γηΐηπρ nf 6 mb mn rnnplny^rl Ar this time, 0.1 M o-methyl-mannoeide in selection buffer was then added, followed by a 4 mL total volume wash. Thrombin enzymatic activity was detected via chromogenic substrate monitored by absorbance at 405 nm. Peak thrombin fractions were pooled, extracted with phenol, and ehe volume reduced by nBuOH extraction. 20 pg glycogen was added, the single-stranded 98-mer precipitated via ethanol addition and pelleted via centrifugation, The pelleted DNA was resuspended in water and used as a template for PCR amplification. This protocol was repeated to obtain a pool of DNA that resulted from 5 rounds of sslsction on thrombin columns.

Double-stranded DNA was digested with BcoRI and HinDIII and cloned into pOBM3Z. Aptamers were then transformed into 1. coll and analyzed by dideoxy sequencing. Round five aptamer pool DNA bound to thrombin with a Rg of approximately 300 nM.

SXflmplfl 11 Demonetration of Aptamer Specificity for Binding 2S to and Inhibition og Thrombin The specificity of aptsmer binding was demonstrated using 33P radiolabeled DNA and a series of proteins. To determine the binding specificity of the thrombin aptamer, 9<-mer clone *29, having the partial 30 sequence 5'COOQQA3AGGTTGGTGTGGTTQQCAATGGCTAGAGTAGTOAC GTTITOGCQQTGAflQTCC 3' wae used. The consensus sequence ls shown underlined. In addition, a 2l-mer aptamer, s* (κ>π«χκπΏαττοοατΓαοο 3* was tested for inhibition of another fibrinogen-cleaving enzyme ancrod, which was 35 obtained commercially (Sigma, Cat. No. A-5042). The .55. 21-mar had a of K2 for thrombin of about 100 nH and ita Kjj was about 350 nN. Clone *29 had a 1¾ of about 200 nM for thrombin.

The aptamer wae shown to specifically bind to 5 thrombin by a filter binding assay. Briefly, radiolabeled aptamer DNA at about a concentration of about 1 nM was incubated with the Indicated protein for several minutes at room teuperature, followed by filtration of the aptamer-protein mixture through a nitrocellulose filter. The filter was washed with 3 mL of selection buffer and then radioactivity bound to the filters was determined as a * of input radioactivity. Results obtained are shown in Table 7. Binding data is shown for both unselected 96-mer DNA and for two separate experiments with clone *29 96-mer. All proteins were tested at a ΙμΜ concentration except human serum albumin which was used at 100 μΜ. The results that were obtained demonstrated that the 96-mer specifically bound to thrombin and had little affinity for most of the other proteins tested. -56* Table 7 Protein Unm acted-pha input CPM fl2und_cai 3 Bound 5 Control 75573 230 0 --- Throobin 74706 6732 9.0 Prothrombin 7S366 183 <0.5 Albumin 76560 1851 2.0 Chymotrypsin 75566 235 <0.5 10 Trypsin 73993 306 <0.5 Kallikrein 76066 122 <0.S Plasmin 74513 3994 5.0 15 Clone 29 DNA Control 81280 126 0 Throobin 81753 48160 59.0 Prothrombin 81580 8849 11.0 Albumin (100 μΜ) 85873 1778 2.0 Chymotrypsin 82953 207 <0.5 20 Trypsin 75673 318 <0.5 Kailikrsin 84013 143 <0.5 Plasmin 82633 12323 15.0 TPA 81960 192 <0.5 25 Clone 39 DMA Control 81886 917 0 Thrombin 82940 48796 59.0 Prothrombin 91760 8719 9.5 Albumin 92473 234 <0.5 30 Chymotrypsin 97060 186 <0.5 Trypsin 97846 429 <0.5 Kallikrein 95053 1275 <0.5 Plasmin 66565 9704 15.0 TPA 98166 644 <0.5 -57The thrombin 21 -mer aacrod assay was conducted as follows. Ancrod was suspended in sterile water at a concentration of 44 U/raL. 10 i& ancrod solution was added to 95 gL of selection buffer prewarmed to 37*C. 100 μΐι of this mixture was transferred to the coagulation cup of the fibrometer described above, followed by addition of 200 μΐι of fibrinogen and 20 μΐι of 2l-mer DNA (both prewarmed to 37*C). TB buffer pH 7.0 was used ae a control lacking DNA. The control clot time was 25 seconds while the clot time in the presence of 500 nM 21mer was 24 seconds and was 26 seconds in the pretence of 33 μΜ 2l-mer. This result demonstrated the specificity on inhibition of fibrinogen cleavage waa limited to thrombin; ancrod waa not affected.

BxBBPlB 12 Thrombin Aptamer Pharmacokinetic Studies A 15-mer single-stranded deoxynucleotide, ' GG1TGGTGTGGTTGG 3*, identified as a consensus sequence from 30 thrombin aptamer clones as described in Bxample 1 above, was used. Young adult rats of mixed gender and strain wars used. The animals were anaesthetised and a diester of the 15-mer was injected through a catheter in 200 μΐ volumes (in 20 mM phosphate buffer, pH 7.4, 0.15 M NaCl) at two concentrations, so that the final concentration of IS-mer in the blood was about 0.5 and 5.0 μΜ respectively, although the exact concentration depends on the volume of distribution (which is unknown for this oligonucleotide). These values are 10 to 100 times greater than the human in vitro value. No heparin waa used for catheterization.

At 0, 5, 20 and 60 minutes, blood wae withdrawn from the animals (approx. 500 μΐ aliquots), transferred into tubes containing o.l volume citrate buffer, and centrifuged. Rat plasma was removed and tested in a 58thrombin clotting-time assay. Six animals wars used at each concentration, and three animals were injected with the control carrier solution containing no 15-mar. λ prolonged clotting time was observed at tha 5 5 minute time point at both concentrations, with ths most significant prolongation occurring at tbs higher dose concentration. Little or no activity was observed at 20 minutes. Thus, ths 15-mer is blood withdrawn from rats 5 minutes post-inject ion was able to inhibit exogenously added human thrombin. A separate APTT test at the 5 minute time point showed that the 15-msr also inhibited rat blood coagulation, presumably by inhibiting rat thrombin to a significant degree. The half-life of the -mer in rats appears to be about 5 minute· or less.

Bxaapla ia Thrombin Aptamer Primate Studies Two thrombin aptamers were administered to adult male cynomologous monkeys. Unsubstituted 15-msr DNA with ths sequence 5' σαττοατατοοττοο 3' and an analog, 5' OOTTGOTOTOOTT*0*0 3', containing thloate internucleotide linkages at ths indicated positions (*), wars used. Aptamer was delivered as an intravenous bolus or infusion and then blood samples wars withdrawn at various times after delivery of ths bolus or during and after Infusion. The catheter was hsparinissd after the 10 minute timepoint. The animals were not systematically heparinized.

Thrombin inhibit ion was measured by a prothrombin time test (PT) ueing · commercially available kit, reagents and protocol (Sigma Diagnostics, St. Louis, catalog Nos. T 0263 and 870-3). Inhibition of thrombin was indicated by an increased clot time compared to the control in the PT test. Clot times were obtained by withdrawing a sample of blood, spinning out red cells and -59using the plasma In tha PT teat. Control thrombin PT clot time value· were obtained several minutes prior to administration of aptamer. Briefly, tha PT assay was conducted using 0.1 mL of monkey plasma prewarmed to 37» C and 0.2 mL of a 1:1 mixture of thromboplastin (used according to manufacturers instructions) and CaCl2 (25 mM), also prewarmed to 37«C. Thrombin clot times were measured with a fibrometer as described above.

The animals were at least two years old and 10 varied in weight from 4 to 6 kg. Doses of aptamer were adjusted for body weight. Aptamer DNA was dissolved in sterile 20 mM phosphate buffer (pK 7.4) at a concentration of 31.8 to 33.2 mg/mL and diluted in sterile physiological saline prior to delivery. Bolus injections were administered to give a final concentration of 22.5 mg/Xg (1 animal) of the diester aptamer or 11.25 mg/Xg (1 animal) of the dleeter aptamer. Infusions were administered over a l hour period to three groups of animals: (i) 0.5 mg/kg/min of dieeter 15-mer (4 animals), (ii) 0.1 mg/kg/min of dieeter 15-mer (2 animals) and (iii) 0.5 mg/kg/min of thioate analog 15mer (2 animals). pt assay results from the bolus injections showed thrombin inhibition times of 7.8, 3.3 and 1.35 times control at 2.5, 5.0 and 10.0 min respectively after delivery of the aptamer for the high dose animal. Inhibition times of 5.4, 2.2 and 1.2 times control were obtained from the low dose animal at the seme time pointe.

Figure 2 shows a plot of the PT times from the animals that received the high dose dieeter infusion compared to pretreatment control values. The data pointe show the PT clot time ae an average value obtained from the 4 animals in the group. The arrows Indicate time pointe at the beginning and end of the infusion period. -60Thrombin inhibition peaked at about 10 to 20 min after the infueion wae initiated and remained level until the infusion period was terminated. Inhibitory activity decreased rapidly after the Infusion of aptamer terminated.

High dose diester and high dose thioate animals showed comparable inhibition of thrombin-mediated clotting, with the high dose thioate giving a sustained clot time of 3.5 to 2.7 times the control value during the course of the infusion. The low dose diester compound gave a clot time of 1.4 to 1.5 times the control value. These results demonstrated the efficacy of the native and thioate analog aptamers in primates.

Example 14 Inhibition of Extracorporeal Blood Clotting Bv Thrombin Aptamer Anticoagulation of a hemodialysis filter was demonstrated using the 15-mer 5' GGTTOGTGTGGTTOG 3' thrombin aptamer with human blood. A bolus of 15-mer DNA was delivered to human blood at 37C to give aa aptamer concentration of ΐΟμΜ. The blood was contained in an extracorporeal hemodialysis circuit (Travenol, Model No. CA-90). Pressure proximal to the hemodialysis filter was monitored to determine the time after administration of aptamer that coagulation occurred. Blood coagulation wae marked by a pressure increase from about 50 ma Hg observed with uncoagulated blood (blood flow rate 200 mL/min) to pressure of at least 400 mm Hg.

Using untreated blood, coagulation occurred at about 9 minutes after fresh blood wae placed ln the hemodialysis unit and circulation was begun. A heparin control (l U/mL) gave sustained anticoagulation until the experiment wae terminated at 60 minutes after start of circulation ln the unit. Blood coagulation occurred at IE 920551 minutes in one trial with ths 15-mar. In a second trial, coagulation did not occur during ths 60 minut· courts of the experiment.

Thus, methods for obtaining aptamere that specifically bind serum proteins such as thrombin and Factor X, eicosanolds, kinins such as bradyklnin, and call surface ligands ars described, ae well as the therapeutic utility of these aptamers and ths use of ths aptamers in the detection end isolation of such substances. Although preferred embodiments of ths subject invention have been described in some detail, it is understood that obvious variations can bs mads without departing from ths spirit and scope of the appended claims.

Claims (5)

1. We claim: 1. An aptamer containing a binding region capable of binding specifically to throcnbin.

2. The aptamer of claim l wherein the aptamer ie eelected from the group coneieting of single-stranded RNA, single-stranded DNA, double-stranded dna and chemical modifications thereof.

3. The aptamer of claim 2 wherein the aptamer is single-stranded RNA.

4. The aptamer of claim 2 wherein the aptamer 15 is single-stranded DNA.

5. The aptamer of claim 2 wherein the aptamer is double-stranded DNA. 20 6. An aptamer containing at least one binding region capable of binding specifically to thrombin with a dissociation constant (Kd) of less than 30 χ 10’ 3 . 7. The aptamer of claim 6 containing at least 25 one binding region capable of binding specifically to thrombin with a dissociation constant JXd) of less than io* 3 . t 8. An aptamer containing at least one binding 30 region capable of binding specifically to thrombin wherein said binding region contains less than 16 nucleotide residues. 9. The aptamer of claim 8 wherein said binding 35 region contains between 6 and 15 nucleotide residues. -S310. Tho aptamer of claim 1 wherein the aptamer contains at leaet one modified base, sugar, or linking group. 11. The aptamer of claim 10 wherein the aptamer contains at least one linking group wherein P(0)0 le replaced hy P(O)S, P(8)8, p(O)NR 2 , P(O)R, P(O)OR', CO or CK 2 , wherein each R or R* ia 10. Independently H or substituted or uneubetltuted alkyl (1-20C) optionally containing an ether (-0-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl; or the aptamer contains at least one linking group attached to an adjacent nucleotide through 8 or N; or 15 the aptamer contains at least one analogous fonn of purine or pyrimidine, or at least one abaalc site. 11. 12. The aptamer of claim 11 which ie a single20 stranded DNA. 12. 13. The aptamer of claim 11 which contains at least one linking group wherein F(0)0 is replaced by P(O)S, and wherein said linking group is attached to each 25 adjacent nucleotide through 0. 13. 14. The aptamer of claim 11 which contains at least one linking group wherein P(0)0 is replaced by P(0)NH(CK 2 CK 2 OCM 2 ), and wherein said linking group is 30 attached to each adjacent nucleotide through 0. 14. 15. The aptamer of claim 11 which contains at least one linking group wherein P(O)O is replaced by CK 2 , and wherein said linking group is attached to each 35 adjacent nucleotide through 0. -6416. The aptamer of claim ll wherein the aptamer ie single- or double-stranded DMA and contains at least one uracil (dU) bass substituted for thymine. 17. The aptamer of claim 11 containing at lsaet one 5-pentynyluracil base substituted for thymine. 15. 16. The aptamer of claim 11 containing at 10 least one abasic site. 16. 19. An aptamer capable of binding specifically to thrombin wherein the aptamer contains at least one modified or analogous sugar. 17. 20. The aptamer of claim 19 wherein the at least one modified or analogous sugar is a furanose sugar. 20 18. 21. The aptamer of claim 20 wherein the furanose sugar is a 2'-modified furanose sugar. 19. 22. The aptamer of claims 1-21 wherein said binding region coaprises the sequence QQXTOQ, wherein X 25 le T, A, U, dU or 0. 20. 23. The aptamer of claim 22 wherein said nucleotide sequence has the formula OOTTGQ. 30 21. 24. The aptamer of claim 22 wherein said thrombin binding region conprises the eequence OQXTOQ (N) ^GOXTOG or a fragment thereof, wherein N is O, A, C, U, dU or T, and ι ia an integer from 2 to 5. •65* 22. 25. The aptamer of claim 24 wherein said sequence has the formula OQTtgotgtggttoo. 23. 26. The aptamer of claim 25 having the formula 5 ΟΟΊΤΟΟΤΌΤΟΟΤΤΟΟ*θ·τ wherein * denotes an MBA linkage. 24. 27. The aptamer of claim 25 having the formula GGTTGGTGTGGTT*G*G wherein * denotes a thioate linkage. 10 25. 28. The aptamer of claim 25 having the formula G*G*T*T*G*G*T*G*T*G*G*T*T*G*G wherein * denotes a thioate linkage. 26. 29. The aptamer of claim 25 having the formula 15 GGTTGG(dU)Q(dU)GGTTGG. 27. 30. The aptamer of claim 25 having the formula GG (dU) TGGTGTGQ (dU) TGG. 20 28. 31. The aptamer of claim 25 having the formula GGTTGGTGTGGTU'GG wherein u* denotes 5-pentynyluracil. 29. 32. The aptamer of claims 1-23 which contains a binding region of lees than 16 nucleotide residues. 30. 33. The aptamer of claims 1-21 which contains a binding region of lees than 6 nucleotide residues. 31. 34. The aptamer of claims 1-31 which contains 30 6-100 nucleotide residues. 32. 35. The aptamer of claim 34 which contains 650 nucleotide residues. 6636. The aptamer of claims 1-35 wherein said aptamer is capable of binding specifically to thrombin at physiological conditions. 37. aptamer binds 10·’. The aptamer of claims 1-35 wherein said to said target with a Kd of less than 30 x aptamer 10·’ at 33. 38. The aptamer of claim 37 wherein said binds to ths target with a Kd of less than 30 x physiological conditions. 34. 39. The aptamer of claims 1-38 wherein the Kd with respect to the aptamer and thrombin is less by a 15 factor of at least 10, as compared to ths Kd for said aptamer and other molecules. 35. 40. The aptamer of claims 1-39 whieh is a secondary aptamer. 36. 41. λ method for obtaining an aptamer containing at least one binding region that specifically binds thrombin, which method comprisesi (a) incubating thrombin with a mixture of 25 oligonucleotides under conditions wherein complexation occurs with some, but not all, members of the mixture to form oligonucleotide-thrombin cooplexes; (b) separating the oligonucleotide-thrombin complexes from uncomplexed oligonucleotide; 30 (c) recovering and anplifying the complexed oligonucleotide from said complexes; and -6742. The method of claim 41 wherein said aptamer ia a single-stranded DNA, or wherein said aptamer contains at least one binding region capable of binding specifically to 5 thrombin with a dissociation constant (Kd) of less than 30 x IO’’, or wherein said aptamer contains at least one binding region capable of binding specifically to thrombin wherein the Kd with respect to the aptamer and 10 thrombin ie less by a factor of at least 10, as compared to the Kd for said aptamer and other molecules, or wherein said aptamer contains at least one binding region capable of binding specifically to thrombin wherein said binding region contains less than 15 16 nucleotide residues. 37. 43. The method of claim 42 wherein said mixture of oligonucleotides contains at least one modified oligonucleotide. 38. 44. The method of claim 42 wherein said amplifying is conducted ueing at least one modified nucleotide. 25 39. 45. Tha method of claims 42*44 wherein eald mixture of oligonuoleotidee contains at least one randomised-sequence region. 40. 46. The method of claims 42-45 which further 30 Includes repeating steps (a) * (c) ueing the recovered and amplified complexed oligonucleotidee resulting from step (c) in succeeding step (a). 41. 47. The method of claime 42-46 wherein the 35 binding affinity oligonucleotide mixture for thrombin ie • ββat least 50«fold lees than the binding affinity of the aptamer for thrombin. 42. 48. An aptamer prepared by the method of 5 claims 42-47. 43. 49. A method to obtain a secondary aptamer for thrombin which method comprises: (a) incubating thrombin with a mixture of 10 oligonucleotide sequences under conditions wherein complexation occurs with some, but not all, members of the mixture to form oligonucleotide-thrombin complexes; (b) separating the oligonucleotide-thrombin complexes from uncomplexed oligonucleotides; 15 (c) recovering and amplifying the complexed oligonucleotides from said complexes; (d) optionally repeating steps (a)-(c) with the recovered oligonucleotides of step (c); (e) determining the sequences of the recovered 20 oligonucleotides; (f) determining a consensus sequence Included in the recovered oligonucleotides; and (g) synthesizing a secondary aptamer which comprises the consensus sequence. 44. 50. A secondary aptamer prepared by the method of claim 45. 45. 51. A complex formed by thrombin and the 30 aptamer of claims 1-40« 48, or 50. 46. 52. A method for obtaining an aptamer containing at least one binding region that specifically binds thrombin, which method cosprisesi -69(a) incubating thrombin with a mixture of oligonucleotide· under condition· wherein complexation occurs with some, but not all, members of the mixture to form oligonucleotide-thrombin complexes; 5 (b) separating the oligonucleotide-thrombin complexes from uncomplexed oligonucleotide; (c) recovering and amplifying the complexed oligonucleotide from said complexes; and (d) optionally determining the sequence of the 10 recovered oligonucleotide, wherein the dissociation constant (Kd) with respect to said target and mixture of oligonucleotides is l μΜ, or wherein the Kd with respect to the aptamer and 15 said target is less by a factor of at least 50 as compared to the Kd for said target and said mixture of oligonucleotides; or wherein steps (a) and (b) are conducted under physiological conditions, or 20 wherein said mixture of oligonucleotides consists of single-stranded DNA. 47. 53. The method of olaim 52 wherein said mixture of oligonucleotides contains at least one 25 modified oligonucleotide. 48. 54. The method of claim 52 wherein said amplifying is conducted using at least on· modified nucleotide. 49. 55. The method of claims 52-54 wherein said mixture of oligonucleotides contains at least one randomized-sequence region. -7056. Th· method of elaime 52-55 which further includes repeating steps (a)-(c) using tbs recovered and amplified complexed oligonucleotides resulting from step (c) in succeeding step (a). s 50. 57. An aptamer prepared by the method of claims 52-56. 51. 58. A method to detect the presence or absence 10 of thrombin, which method comprises contacting a sample suspected of containing thrombin with the aptamer of claims 1-40 under conditions wherein s complex between thrombin and the aptamer is formed, and detecting the presence or absence of said 15 conplex. 52. 59. A method to purify thrombin, which method comprise· contacting a sample containing thrombin with the aptamer of claims 1-40 attached to solid support 20 under conditions wherein thrombin is bound to ths aptamer coupled to solid support) washing unbound cooponents of the sample) and recovering thrombin from said eolid support. 25 53. 60. A pharmaceutical composition for medical use conprlslng ths aptamer of claims 1-40 in admixture with a physiologically acceptable excipient. 54. 61. A composition for diagnostic uss which 30 coaprises ths aptamer of claims 1-40. 55. 62. The aptamer of claims 1-40 coupled to an auxiliary substance. -7163. The aptamer of claim 62 wherein aald auxiliary aubetanee ie selected from the group consisting of a drug, a toxin, a solid support, and specific binding reagent, a label, a radioisotope, or a contrast agent. S 56. 64. The aptamer of claim 63 wherein said auxiliary substance is a radioisotope selected from the group consisting of lw X, ,,B Tc, *®Y, lll In and 1M l. 10 65. A method to obtain an aptamer containing a binding region which specifically binds thrombin which comprises ι (a) incubating thrombin reversibly coupled to a support with a mixture of oligonucleotide sequences under 15 conditions wherein the coupled thrombin complexes with some, but not all, members of the mixture to form support-hound oligonucleotide complex··; (b) decoupling and recovering the oligonucleotide-thrombin complex from the support to 20 obtain free aptamer-thrombin complexes; (c) recovering and amplifying the complexed oligonucleotides from the free oligonucleotide-thrombin complexes to obtain a population of aptamers; (d) optionally repeating steps (a)-(c) using as 25 said mixture the recovered population of aptamere of step (c); and (·) optionally determining the sequence of the recovered aptamers. 30 66. Ths method of claim 65 wherein the support is a lectin support. 67. The method of claim 66 wherein in step (b), decoupling is accomplished by adding a 35 monosaccharide. -7268. The method of claim 67 wherein tha monosaccharide is a-methyl-mannoside. 5 69. The method of claim 68 wherein the support is a concanavalln λ column. 70. A composition for use in inhibiting clotting or coagulation in a patient which conpoeition 10 conprises an aptamer ae described in claims 1-40. 71. λ composition for use in inhibiting or reducing restenosie in a patient, which compoeition comprises an aptamer as described in claims 1-40. 72. A conpoeition for use in treating a patient's blood ax corpora to inhibit clot formation, which composition comprises an aptamer as described in claime 1-40. 73. A method to prevent coagulation during cardiopulmonary bypaee surgery, which method comprises contacting blood with an aptamer ae described in claims 1-40. 74. In a method eo inhibit clot formation in a patient which comprieee administering to eaid patient a fibrinolytic agent selected from the group coneieting of tissue plasminogen activator and etreptokinaee, the 30 improvement which comprises: coadministering to eaid patient an aptamer ae described in claime 1-40. -7375. An aptamer according to claim 1, substantially as hereinbefore described and exemplified. 76. A method for obtaining an aptamer according to claim 1 or 6, substantially as hereinbefore described and exemplified . 77. An aptamer according to claim 1 or 6, whenever obtained by a method claimed in any one of claims 41-47, 52-56, 57. 65-69 or 76. 78. A method according to claim 49 to obtain a secondary aptamer for thrombin, substantially as hereinbefore described and exemplified. 79. A secondary aptamer for thrombin, whenever obtained by a method claimed in claim 49 or 78. 80. A complex according to claim 51, substantially as hereinbefore described and exemplified. 81. A method according to claim 58, substantially as hereinbefore described and exemplified. 82. A method according to claim 59 to purify thrombin, substantially as hereinbefore described. 83. Thrombin whenever obtained by a method claimed in claim 59 or 82. 84. A pharmaceutical composition according to claim 60, substantially as hereinbefore described. -7485. as hereinbefore A composition according described. to claim 61, substantially 86. An aptamer according to claim 62, substantially as hereinbefore described. 87. A composition according substantially as hereinbefore described. to any one of claims 70-72,