BACKGROUND OF THE INVENTION A. Hemostasis Hemostasis is initiated when a blood vessel suffers trauma, and is characterized by the rapid deposition of platelets and a fibrin clot at the site of an injury. The platelets orchestrate vasoconstriction and the repair of the blood vessel, and help form a hemostatic plug. The insoluble fibrin clot is the major component of the blood clot, and provides an anchoring framework for the platelets.

The fibrin clot forms under the control of at least two convergent blood coagulation pathways, termed the intrinsic and extrinsic pathways. The pathways comprise a cascading series of reactions involving more than ten plasma factors. At each step in the coagulation pathways, an inactive plasma factor is activated through proteolytic cleavage by one or more upstream plasma factors, and the newly activated plasma factor (often in a complex of other co-factors) then activates a downstream plasma factor. This results, ultimately, in the activation of the serine protease thrombin from the inactive precursor prothrombin; thrombin then cleaves fibrinogen to form fibrin. In blood clotting nomenclature, each inactive plasma factor is numbered with a Roman numeral, e. g., Factor V, and the suffix"a"is appended to denote the activated form of each factor (e. g., Factor Va). Many of the plasma factors can occur both free in plasma (plasma-derived factors), or can be associated with the surface of platelets (platelet-derived factors). In some instances, an individual factor can have different properties depending on whether it is in the plasma-derived form or

the platelet-derived form. Such differences may result from differing post- translational modifications.

The catalytic nature of the series of reactions, and the presence of positive- feedback loops, endows the system with great sensitivity. It allows an initially small molecular response to trauma to be rapidly amplified, leading to the formation of a macromolecular clot.

The blood coagulation system must be carefully and dynamically regulated to ensure that the response to trauma is appropriate. If the blood clotting is inefficient, then hemorrhage can occur; conversely, if the clotting system is too active, then thrombosis results. Furthermore, as the injured blood vessel is repaired, a delicate balance must also be reached between the fibrinogenic coagulation pathways and the fibrinolytic pathway. This insures that an effective hemostatic seal is present only for a period sufficient to allow endothelial regeneration to take place.

If the system is deficient in the activity of any one of a number of plasma factors in the cascade, then clotting is inefficient, or even blocked, and hemorrhage results. Conversely, if one of the plasma factors has an inappropriately high or unregulated activity, then thrombosis can result. To prevent thrombosis, many of the factors are negatively regulated, allowing their activity to persist only for as long as is needed to form a clot of the appropriate size. This negative regulation is achieved through the action of proteases, inhibitory-factors, negative-feedback loops, and also simply through rapid dilution of factors from the site of trauma by blood flow.

B. Activated Protein C Activated protein C (APC) is a key negative regulator of the blood clotting system. This potent anti-coagulator inactivates the plasma-derived forms of Factor Va and Factor Villa by proteolytic cleavage, thereby indirectly reducing the activity of the terminal enzyme in the clotting pathway, thrombin. APC is a serine protease synthesized in the liver, and is found on the surface of endothelial cells as an inactive precursor. The inactive precursor is transiently activated by a complex of thrombin and thrombomodulin, and is inactivated by specific inhibitors. The action of these

inhibitors serves to rapidly remove APC activity from the bloodstream. In vitro, APC functions as a potent anti-coagulator only in the presence of membranous material. In vivo, inherited mutations in APC lead to a fatal infant thrombosis termed purpura fulminans.

APC cleaves plasma-derived Factor Va between the arginine residue at position 506 and the glycine residue at position 507, and also between the arginine residue at position 306 and the asparagine residue at position 307. Cleavage at these sites results in complete inactivation of the plasma-derived form of Factor Va.

Further APC cleavage sites have been described between arginine 679 and lysine 680, and around lysine 994, but their function in Factor Va inactivation in vivo is not clear.

Factor Villa is structurally and functionally similar to Factor Va, and is believed to be cleaved by APC between arginine 562 and glycine 563, between arginine 336 and methionine 337, and between arginine 740 and serine 741. For both Factor Va and Factor VIIIa, cleavage at the APC cleavage sites prevents Factor Va and Factor Villa from activating their downstream targets. Factor VIIIa serves as a cofactor in a complex that activates plasma Factor X. The resulting plasma Factor Xa forms a complex with Factor Va that activates prothrombin to form thrombin. The existence of a positive feedback loop can clearly be seen at this level in the pathway, as thrombin then promotes the further activation of Factor V and Factor VIII.

C. Inherited Thrombosis Deep vein thrombosis (DVT) can occur when the blood clotting system is unregulated. Unlike an arterial thrombosis, the formation of a DVT is not normally triggered by an injury to a blood vessel, nor does it involve the participation of platelets. An embolus formed as a result of DVT, often in a vein draining a leg muscle, can travel through the bloodstream until it becomes wedged in an artery, causing an embolism. If an embolism is formed in pulmonary tissue, cardiac muscle, or brain tissue, the resulting tissue necrosis can lead to severe disability or death. Risk factors for DVT include long term immobilization, recent surgery, the use of oral contraceptives, and pregnancy. In addition, inherited disorders of the blood clotting system may also predispose towards thrombosis.

The response of plasma-derived Factor Va to APC is a major determinant in the pathogenesis of venous thrombosis. The blood of between 50% and 60% of all individuals with a family history of thrombosis manifests resistance to the anti- coagulant properties of APC in vitro. In the vast majority of these individuals, APC resistance results from a common inherited defect in the Factor V gene. This mutation, a A-G substitution, leads to the substitution of the arginine residue at position 506, which is one of APC cleavage sites, with glutamine. The resulting aberrant factor, termed Factor V Leiden, functions normally in the clotting pathway, but the plasma-derived form is not cleaved, and hence not inactivated, by APC. The persistence of Factor Va pro-coagulation activity leads to an 80-fold increase in the probability of experiencing venous thrombosis in individuals homozygous for the Leiden mutation. This translates to the expectation of experiencing at least one venial thrombotic episode during a typical lifespan. This probability may be increased when certain risk factors are present, such as immobilization, pregnancy, the use of oral contraceptives, and recent surgery.

It is believed that between 2%-5% of people of European ancestry are heterozygous carriers of the Factor V Leiden mutation. This is associated with a seven-fold increase in the chance of suffering of a deep-vein thrombosis, which may in turn lead to an increased chance of suffering a coronary or a pulmonary embolism.

Carriers of this mutation may also experience elevated rates of second-trimester miscarriages. It is likely that mutations associated with the position 306-307 APC cleavage site in Factor Va, and with the Factor Villa APC cleavage sites, will also play a role in other non-Leiden thrombophilic diseases.

Given the remarkably high incidence of the Factor V Leiden mutation, it is desirable to have a quick and economical method for detecting this mutation.

Diagnosed carriers of the mutation can then act to remove risk factors, or they can be treated prophylactically with anti-coagulants such as coumarin derivatives if it is not possible to remove an important risk factor, e. g., if the carrier is pregnant, or must undergo surgery. Furthermore, it would also be desirable to have a method that can easily be adapted for detecting other mutations in Factor Va and Factor VIIIa that may also give an APC resistant phenotype. Such mutations may, as in Leiden, directly

affect the APC cleavage site (s), or they may interfere with the overall structure of the protein thereby interfering with the ability of APC to bind specifically to the factors.

D. The Current State of the Art in Factor V Leiden Detection Currently, a time-consuming and labor-intensive two step methodology is used to test individuals with a family history of thrombosis for the presence of the Leiden mutation. The first step is an assay to determine whether the individual is APC resistant. This functional assay involves measuring the period required for clot formation in recalcified blood plasma after contact activation and addition of platelet substitutes. The test, termed aPTT (activated partial thromboplastin time), is conducted in the presence and absence of exogenous APC. If the aPTT in the presence of exogenous APC is not more than two fold shorter than in the absence of APC, then APC resistance is diagnosed. Determining the precise time of anti- coagulation requires that highly skilled operators monitor the blood samples; hence, this assay is not amenable to high throughput automated analysis.

Following the diagnosis of APC resistance, the Polymerase Chain Reaction (PCR) is performed using primers that specifically amplify the Leiden mutation. This PCR assay involves isolation of DNA from the buffy coat, performing the PCR, electrophoresing the amplified products, and comparing the sizes of the amplified products with those obtained from a wild-type individual and a Factor V Leiden homozygote. The relatively high cost of the Polymerase Chain Reaction, both in terms of reagents and labor time, necessitates performing the preliminary aPTT test.

One major drawback of this approach is that the functional nature of the aPTT test makes it unsuitable for patients taking anti-coagulant drugs, such as heparin and warfarin. These drugs typically inhibit the final steps of clot formation, namely the cleavage of prothrombin to thrombin by Factor Xa, and the cleavage of fibrinogen by thrombin to form the insoluble fibrin clot.

A further drawback of this functional test is that many other substances can interfere with blood clotting. For example, the presence of lupus anticoagulant, liver disease, and deficiencies in other blood clotting factors will all give rise to an abnormal aPTT result. Furthermore, the use of a number of common medications,

such as oral contraceptives and aspirin-containing pain killers (which have anti- coagulant properties), can also interfere with the aPTT measurement. All of these substances interfere with stages in the coagulation pathway distinct from those directly involving Factor V. Because the aPTT test simply measures coagulation time, it is impossible to determine whether an abnormality in the response to APC is due to a defect in Factor V, or due to the action of these substances on other upstream or downstream stages in the pathway. Conversely, the presence of agents that inhibit coagulation steps downstream from Factor Va may mask a true deficit in Factor Va function. Thus, an unambiguous diagnosis of Factor V Leiden can only be made following the PCR assay.

It has been suggested that in order to get a true measurement of APC activity with respect to Factor V in a functional assay, it is necessary to perform the aPTT test by diluting the patients blood into a carefully selected control blood sample from which all Factor V has been removed by immunoprecipitation. The rationale is that the control blood contributes all of the coagulation factors except the Factor V, and the true effect of APC on Factor V can be studied in a controlled manner. However, an obvious drawback of this approach is that the absolute value of APC resistance will vary with the source of control blood. Furthermore, it has been shown that patients with lupus anticoagulant had a score in this modified test very similar to Leiden heterozygotes. Finally, it is obvious that using blood that is immunodepleted will raise the cost of the test even further.

Yet another drawback of the aPTT test is that it may not be used in blood samples that have been placed in glass tubes treated with an anti-coagulant such as heparin (blood normally coagulates when in contact with a glass surface). This means that blood samples must be frozen prior to assaying, which can interfere with the hemostatic system and therefore with the accuracy of the test.

Other tests have been proposed to assay for deficiencies in the APC-mediated cleavage of plasma Factor Va and Factor VIIIa. These assays all use monoclonal antibodies against specific cleavage fragments of these plasma factors. Such assays, again, are wholly unsuitable for large-scale screening of populations. In order to perform such an assay, the plasma factors must be isolated from blood, subjected to

denaturing polyacrylamide gel electrophoresis, followed by Western blotting of the gel and detection of the blotted plasma factor fragments using labeled secondary antibodies. In such assays, the reduction or absence of certain cleavage products is taken as evidence of APC-resistance. Those skilled in the art will recognize that this is clearly an even more time-consuming approach than the Polymerase Chain Reaction method.

E. The SELEXTMProcess The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in United States Patent Application Serial No. 07/536,428, filed June 11,1990, entitled"Systematic Evolution of Ligands by Exponential Enrichment,"now abandoned, United States Patent Application Serial No. 07/714,131, filed June 10,1991, entitled"Nucleic Acid Ligands", now United States Patent No. 5,475,096, United States Patent Application Serial No. 07/931,473, filed August 17,1992, entitled"Nucleic Acid Ligands", now United States Patent No.

5,270,163 (see also WO 91/19813), each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEXT Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands, each ligand having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two-and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually

any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.

The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.

It has been recognized by the present inventors that the SELEXT method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.

The present inventors have recognized that SELEX or SELEXTM-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target. In theory, within a candidate mixture of approximately 1013to 1018 nucleic acids, the present inventors postulate that at least one nucleic acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.

The basic SELEXT method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No.

07/960,093, filed October 14,1992, entitled"Method for Selecting Nucleic Acids on the Basis of Structure,"now abandoned (see, United States Patent Application Serial No. 08/198,670, now United States Patent No. 5,707,796), describes the use of the SELEXT process in conjunction with gel electrophoresis to select nucleic acid

molecules with specific structural characteristics, such as bent DNA. United States Patent Application Serial No. 08/123,935, filed September 17,1993, entitled "Photoselection of Nucleic Acid Ligands,"now abandoned (see, United States Patent Application Serial No. 08/612,895, now United States Patent No. 5,763,177), describes a SELEXT based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. United States Patent Application Serial No.

08/134,028, filed October 7,1993, entitled"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,"now United States Patent No.

5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25,1993, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX,"now United States Patent No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.

The SELEXT method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.

Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEXT process-identified nucleic acid ligands containing modified nucleotides are described in United States Patent Application Serial No. 08/117,991, filed September 8,1993, entitled"High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,"now United States Patent No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5-and 2'-positions of pyrimidines. United States Patent Application Serial No.

08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-O- methyl (2'-OMe). United States Patent Application Serial No. 08/264,029, filed June 22,1994, entitled"Novel Method of Preparation of 2'Modified Pyrimidine Intramolecular Nucleophilic Displacement,"now abandoned (see, United States

Patent Application Serial No. 08/732,283), describes oligonucleotides containing various 2'-modified pyrimidines.

The SELEXTM method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXTM,"now United States Patent No. 5,637,459, and United States Patent Application Serial No. 08/234,997, filed April 28,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEXTM,"now United States Patent No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and the efficient amplification and replication properties of oligonucleotides with the desirable properties of other molecules.

The SELEXT method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in United States Patent Application Serial No. 08/434,465, filed May 4,1995, entitled"Nucleic Acid Complexes". VEGF nucleic acid ligands that are associated with a Lipophilic Compound, such as diacyl glycerol or dialkyl glycerol, in a diagnostic or therapeutic complex are described in United States Patent Application Serial No. 08/739,109, filed October 25,1996, entitled"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes."VEGF nucleic acid ligands that are associated with a Lipophilic Compound, such as a glycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound, such as polyethylene glycol, are further described in United States Patent Application Serial No. 08/897,351, filed July 21,1997, entitled "Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes." VEGF nucleic acid ligands that are associated with a non-immunogenic, high molecular weight compound or lipophilic compound are also further described in PCT/US97/18944, filed October 17,1997, entitled"Vascular Endothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes."Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.

SUMMARY OF THE INVENTION The invention described herein provides methods of identifying and producing nucleic acid ligands to the mutated plasma clotting Factor V Leiden. It is an object of the present invention to provide a rapid, sensitive and economical test for the Factor V Leiden mutation that prevents the factor from being inactivated by activated protein C.

This invention further provides a Factor V Leiden mutation detection assay that does not depend on the formation of a blood clot in vitro, and hence can be performed on a) blood from patients currently taking anti-coagulant medications that inhibit the final steps of blood clot formation; b) blood from patients in which other substances affect the formation of a clot ; and c) blood stored in heparin-treated receptacles.

This invention also includes a one-step method for the detection of Factor V Leiden that can be performed routinely and cost-effectively using high-throughput, automated instruments. Also included is a method for detecting resistance to activated protein C in a blood sample without measuring the activated partial thrombin time.

A method for detecting other Factor V mutations, and mutations in other plasma factors, that impart resistance to activated protein C is also included within the scope of the invention.

The central concept of the invention is the use of nucleic acid ligands that recognize and bind to Factor V Leiden at the mutated glutamine 506-glycine 507 APC cleavage site. Some of the nucleic acid ligands of the invention bind to a discrete epitope that comprises this mutated cleavage site and a predetermined number of amino acids both N-terminal and C-terminal to the mutated cleavage site.

In preferred embodiments, ligands to the Factor V Leiden mutated activated protein C cleavage site are used in an assay in which blood is treated with a proteolytic enzyme (s), such as thrombin, in order to cleave all Factor V (wild-type and mutant) to the active form, Factor Va (wild-type and mutant). The blood is also treated with exogenous activated protein C, which cleaves and inactivates only wild- type Factor Va. If the Factor V Leiden mutation is present in the blood sample, then

the nucleic acid ligand will bind to its cognate epitope. This ligand will not cross react even weakly with any wild-type Factor Va present in the sample (resulting from heterozygosity for the mutation), as this will all be cleaved by activated protein C at a site within the epitope. This provides the specificity of the assay. If any of the nucleic acid ligand is found complexed with Factor Va, then it can be unambiguously concluded that the blood sample contains the Factor V Leiden recognized by the nucleic acid ligand.

The invention described herein also provides methods for detecting the binding of the nucleic acid ligand to the Factor V Leiden. In one preferred embodiment, the binding to Factor V Leiden is accompanied by a change in a fluorescence signal in the assay mixture. The magnitude of this signal can be used to determine whether the individual being tested is homozygous or heterozygous for the Factor V Leiden mutation. The invention also provides methods for performing the assay using a solid support-bound nucleic acid ligand. Unlike prior art methods for Factor V Leiden detection, all of the assays described herein are particularly suitable for automated, high-throughput analysis of blood samples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Described herein are methods for producing high affinity ssDNA and RNA ligands to the mutated plasma clotting Factor V Leiden. The central method utilized herein for identifying such nucleic acid ligands is called the SELEXTM process, an acronym for Systematic Evolution of Ligands by EXponential enrichment and involves (a) contacting the candidate mixture of nucleic acids with Factor V Leiden, or expressed domains or peptides corresponding to the Factor V Leiden mutated APC cleavage site, (b) partitioning between members of said candidate mixture on the basis of affinity to Factor V Leiden, and (c) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to Factor V Leiden.

Definitions Various terms are used herein to refer to aspects of the present invention. To aid in the clarification of the description of the components of this invention, the following definitions are provided: As used herein,"nucleic acid ligand"is a non-naturally occurring nucleic acid having a desirable action on a target. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule. In the preferred embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.

Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture ; and c) amplifying the increased affinity nucleic acids to yield a ligand- enriched mixture of nucleic acids.

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

As used herein,"nucleic acid"means either DNA, RNA, single-stranded or double-stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.

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

As used herein,"blood sample"refers to both whole blood and experimentally-derived blood fractions obtained from an individual. This includes plasma fractions from which all cells and fibrin clots have been removed.

"SELEX"methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most strongly with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved. In the present invention, the SELEXT methodology is employed to obtain nucleic acid ligands to Factor V Leiden. The SELEXT methodology is described in the SELEX Patent Applications.

"SELEX target"or"target"means any compound or molecule of interest for which a ligand is desired. A target can be a protein, peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. without limitation. In this application, the SELEXT target is Factor V Leiden. In particular, the SELEX targets in this application include purified Factor

V Leiden, and fragments thereof, and short peptides or expressed protein domains comprising the Factor V Leiden mutation.

As used herein,"activated protein C cleavage site"or"APC cleavage site"is defined as any site at which the serine protease known as activated protein C (APC) cleaves plasma-derived factors Va and Villa. The APC cleavage sites in Factor Va include those located between arginine 306 and asparagine 307, between arginine 506 and glycine 507, and between arginine 679 and lysine 680. A mutated APC cleavage site typically has an amino acid substitution at the amino acid on the N-terminal side of the APC cleavage site. This substitution prevents APC from cleaving at this site.

For example, in the Factor V Leiden mutation, the amino acid at 506 is glutamine rather than arginine.

As used herein,"solid support"is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, magnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide, gold, and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces and grooved surfaces.

As used herein,"ligand beacon"refers to a nucleic acid molecule labeled with a fluorescent group and an adjacent quenching group, that can specifically hybridize to a nucleic acid ligand. Upon doing so, the ligand beacon undergoes a conformational change that causes the quenching group and the fluorescence group to move relative to one another such that the emission from the fluorescent group is modified. Ligand beacons are described in detail in commonly assigned PCT Application No. PCT/US98/26599 entitled"Homogeneous Detection of a Target through Nucleic Acid Ligand-Ligand Beacon Interaction", filed on December 15, 1998. In preferred embodiments, the ligand beacon comprises a stem-loop nucleic acid, wherein the fluorescent group and the quenching group are at the termini of the nucleic acid, and the loop comprises sequences that are at least partially complementary to sequences within the nucleic acid ligand. In some embodiments,

the ligand beacon can only hybridize to the nucleic acid ligand when the nucleic acid ligand is not bound to its target. In other embodiments, the ligand beacon can only hybridize when the nucleic acid ligand is bound to its cognate target. In either case, hybridization of the ligand beacon to the nucleic acid ligand is accompanied by an increase in the fluorescence emission intensity of the ligand beacon. Although in preferred embodiments the ligand beacons are stem-loop nucleic acids, there is no limitation on the three dimensional structure of the ligand beacon. Any structure that can hybridize to a nucleic acid ligand, and in doing so undergo a conformational change that separates initially adjacent nucleotide positions, is contemplated.

Similarly, ligand beacons that undergo conformational changes in which initially separated nucleotide positions become adjacent upon hybridizing to nucleic acid ligands are also included in the invention. These ligand beacons, when labeled with fluorescent groups and quenching groups at the appropriate nucleotide positions, undergo a decrease in fluorescence intensity upon binding to the nucleic acid ligand.

A. Preparing nucleic acid ligands to Factor V Leiden In the preferred embodiment, the nucleic acid ligands of the present invention are derived from the SELEX methodology. The SELEX process is described in U. S. Patent Application Serial No. 07/536,428, entitled"Systematic Evolution of Ligands by Exponential Enrichment,"now abandoned ; U. S. Patent Application Serial No. 07/714,131, filed June 10,1991, entitled Nucleic Acid Ligands, now United States Patent No. 5,475,096 ; United States Patent Application Serial No.

07/931,473, filed August 17,1992, entitled"Nucleic Acid Ligands,"now United States Patent No. 5,270,163 (see also WO 91/19813). These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.

The SELEX process provides a class of products which are nucleic acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired target compound or molecule. Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules. SELEX methodology can also be used to target

biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.

In its most basic form, the SELEX process may be defined by the following series of steps: 1) A candidate mixture of nucleic acids of differing sequence is prepared.

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

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

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.

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

5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specific objectives. For example, United States Patent Application Serial No.

07/960,093, filed October 14,1992, entitled"Method for Selecting Nucleic Acids on the Basis of Structure,"now abandoned (see, United States Patent Application Serial No. 08/198,670, now United States Patent No. 5,707,796), describes the use of the SELEXT process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. United States Patent Application Serial No. 08/123,935, filed September 17,1993, entitled "Photoselection of Nucleic Acid Ligands,"now abandoned (see, United States Patent Application Serial No. 08/612,895, now United States Patent No. 5,763,177), describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. United States Patent Application Serial No.

08/134,028, filed October 7,1993, entitled"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,"now United States Patent No.

5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. United States Patent Application Serial No. 08/143,564, filed October 25,1993, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Solution SELEX," now United States Patent No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. United States Patent Application Serial No. 07/964,624, filed October 21,1992, entitled"Nucleic Acid Ligands to HIV-RT and HIV-1 Rev," now United States Patent No. 5,496,938, describes methods for obtaining improved nucleic acid ligands after SELEXT has been performed. United States Patent

Application Serial No. 08/400, 440, filed March 8,1995, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Chemi-SELEX,"now United States Patent No. 5,705,337, describes methods for covalently linking a ligand to its target.

The SELEXT method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics.

Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEXTM-identified nucleic acid ligands containing modified nucleotides are described in United States Patent Application Serial No.

08/117,991, filed September 8,1993, entitled"High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,"now United States Patent No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5-and 2'-positions of pyrimidines. United States Patent Application Serial No.

08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH2), 2'-fluoro (2'-F), and/or 2'-O- methyl (2'-OMe). United States Patent Application Serial No. 08/264,029, filed June 22,1994, entitled"Novel Method of Preparation of Known and Novel 2'Modified Nucleosides by Intramolecular Nucleophilic Displacement,"now abandoned (see, United States Patent Application Serial No. 08/732,283), describes oligonucleotides containing various 2'-modified pyrimidines.

The SELEXT method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in United States Patent Application Serial No. 08/284,063, filed August 2,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX,"now United States Patent No. 5,637,459, and United States Patent Application Serial No. 08/234,997, filed April 28,1994, entitled"Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,"now United States Patent No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and

replication properties, of oligonucleotides with the desirable properties of other molecules.

The SELEX process provides high affinity ligands of a target molecule.

This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to the specific target of plasma Factor V Leiden.

In commonly assigned U. S. Patent Application Serial No. 07/964,624, filed October 21,1992, methods are described for obtaining improved nucleic acid ligands after the SELEX process has been performed (now United States Patent No.

5,496,938). This patent, entitled"Methods of Producing Nucleic Acid Ligands,"is specifically incorporated herein by reference.

One potential problem encountered in the diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e. g., U. S. Patent Application Serial No. 08/117,991, filed September 9,1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", now abandoned (see, United States Patent Application Serial No. 08/430,709, now United States Patent No. 5,660,985), which is specifically incorporated herein by reference.

Modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such

as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3'and 5'modifications such as capping.

The modifications can be pre-or post-SELEXT""process modifications. Pre- SELEX process modifications yield nucleic acid ligands with both specificity for their SELEX target and improved in vivo stability. Post-SELEX process modifications made to 2'-OH nucleic acid ligands can result in improved in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.

Other modifications are known to one of ordinary skill in the art. Such modifications may be made post-SELEXT process (modification of previously identified unmodified ligands) or by incorporation into the SELEX process.

The preferred nucleic acid ligands of the invention bind to a single epitope in Factor V Leiden. The most preferred epitope comprises the mutated APC cleavage site at position 506-507 and a predetermined number of amino acids on the C-terminal and the N-terminal side of the mutated APC cleavage site.

The nucleic acid ligands of the invention are prepared through the SELEX methodology that is outlined above and thoroughly enabled in the SELEX Patent Applications incorporated herein by reference in their entirety. The SELEX process can be performed using purified Factor V Leiden, or fragments thereof as a target.

Alternatively, full-length Factor V Leiden, or discrete domains of Factor V Leiden, can be produced in a suitable expression system. In preferred embodiments, the cloned domains will comprise the mutated APC cleavage site at position 506-507 and a predetermined number of the amino acids that are present on either side of the mutation. Alternatively, the SELEXT process can be performed using as a target a synthetic peptide that includes the sequence of the APC cleavage site mutation and a predetermined number of the amino acids that are found on either side of these two positions in Factor V Leiden. By way of example only, the Factor V Leiden-specific nucleic acid ligand can be prepared by performing the SELEXT process against a peptide with the same sequence of amino acids as occurs in Factor V Leiden from position 491-521. Determination of the precise number of amino acids needed for the optimal nucleic acid ligand would be a matter of routine experimentation for skilled artisans.

Using SELEX methodology, nucleic acid ligands can be obtained that discriminate between Factor V Leiden and wild-type Factor V, which differ by only one amino acid. This can be done by selecting against nucleic acid ligands in the candidate mixture that have an affinity for the wild-type Factor V or the corresponding region of the wild-type APC cleavage site. This technique of selecting for one or more properties (i. e., selecting for binding to a peptide or expressed domain that includes the Factor V Leiden mutated APC cleavage site), and then selecting against one or more properties (i. e., selecting against binding to the corresponding wild-type APC cleavage site and the products of APC cleavage at this site) is termed Counter-SELEX. Counter-SELEXT is described in detail in United States Patent Application Serial No. 08/134,028, filed October 7,1993, entitled"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,"now United States Patent No. 5,580,737.

Other embodiments of the invention use nucleic acid ligands that bind to regions of Factor V on either side of the APC cleavage site at position 506-507, but do not actually contact Factor V at the position 506 and 507 residues. In one such embodiment, these nucleic acid ligands are obtained by using a peptide that spans the position 506 and 507 APC cleavage site and includes 10 amino acids on either side of position 506 as a SELEX target. Nucleic acid ligands that can bind to this peptide are then subjected to the Counter-SELEXT process to remove nucleic acid ligands that actually bind to the APC cleavage site between position 506 and 507. This can be done using a short peptide that includes the residues at positions 506 and 507, but only includes, for example, 3 amino acids on either side of position 506. The resulting nucleic acid ligands only bind to Factor V that is not cleaved by APC between position 506 and 507.

In some embodiments, the nucleic acid ligands become covalently attached to their targets upon irradiation of the nucleic acid ligand with light having a selected wavelength. Methods for obtaining such nucleic acid ligands are detailed in United States Patent Application Serial No. 08/123,935, filed September 17,1993, entitled "Photoselection of Nucleic Acid Ligands", now abandoned (see, United States Patent

Application Serial No. 08/612,895, now United States Patent No. 5,763,177), which is specifically incorporated herein by reference in its entirety.

B. Assay for Factor Va Activated Protein C cleavage sites Mutants The nucleic acid ligands of the invention can be used in assays for the Factor V Leiden mutation. This mutation disrupts one of the APC cleavage sites of Factor V, thereby preventing the inactivation of this plasma-derived factor by APC, and leads to an increased risk of venous thrombosis. The assay involves the following steps: a) obtaining a blood sample from an individual suspected of carrying the Factor V Leiden mutation; b) contacting said blood sample with: (1) at least one proteolytic enzyme in an amount sufficient to cleave all Factor V present in said blood sample to form Factor Va, (2) activated protein C (APC) in an amount sufficient to cleave all wild-type Factor Va at the appropriate cleavage sites, and (3) a nucleic acid ligand to Factor V Leiden, wherein said nucleic acid ligand binds specifically to an epitope comprising the glutamine residue at position 506, the glycine residue at position 507, and a predetermined number of the amino acids N-terminal to position 506 and C-terminal to position 507 ; c) determining whether said nucleic acid ligand is complexed with Factor Va in said blood sample.

Suitable proteolytic enzymes for use in the cleavage of Factor V to Factor Va include thrombin, Factor Xa, and less specific proteases, such as pronase. In the embodiments that follow, thrombin is used merely as an example of a suitable proteolytic enzyme.

However, it is to be understood that any suitable proteolytic enzyme could also be employed in this invention. All wild-type plasma-derived Factor Va present in the blood sample is cleaved by APC between arginine 506 and glycine 507, thereby rendering it incapable of binding to the nucleic acid ligand which requires an intact APC cleavage site epitope for binding. Factor V Leiden is not cleaved by APC

between positions 506 and 507 due to the arginine-glutamine mutation at position 506 that disrupts the APC cleavage site. Therefore, detection of a complex between the nucleic acid ligand and plasma-derived Factor Va is indicative of the presence of the Factor V Leiden in the blood sample. The lack of cross reactivity of the nucleic acid ligand with wild-type Factor Va following APC cleavage means Factor V Leiden can be detected unambiguously even in individuals heterozygous for the mutation.

Such individuals have both the wild-type and the mutant Va in their bloodstream.

Measurement of the amount of the nucleic acid ligand bound to plasma-derived Factor Va can be used to determine if the individual is homozygous or heterozygous for the Factor V Leiden mutation. The present invention provides methods for performing such allele analysis.

The method of the instant invention is preferably performed on plasma that is devoid of platelets rather than on whole blood. This is because platelet-derived Factor Va is stabilized in an unknown manner against APC inactivation (Carnire, et al.

1998. Blood 91: 2818-2829). Thus, the assay may falsely indicate that Factor V Leiden is present if conducted in the presence of platelets. Platelets and other blood cells can be easily removed from whole blood by centrifugation.

In other embodiments, the nucleic acid ligand recognizes regions of Factor V on either side of positions 506 and 507, but does not actually contact Factor V at positions 506 and 507. Such nucleic acid ligands are described in the preceeding section. If plasma-derived Factor V is cleaved by APC between positions 506 and 507, then the two regions of Factor V to which the nucleic acid ligand binds are separated, and so no binding is possible. In the presence of APC and thrombin, these nucleic acid ligands can only bind to Factor V Leiden. Assays using these nucleic acid ligands are conducted as described above.

In other embodiments, the nucleic acid ligand will recognize other mutated APC-cleavage sites in both Factor Va, and in the homologous plasma clotting factor Factor Villa. Such as yet uncharacterized mutations would be expected by those familiar with the field to give the same phenotypes of APC-resistance and thrombosis as the Leiden mutation, and may indeed account for the remaining 20% or so of thrombotic-individuals with non-Leiden APC resistance. Thus, the methods and

reagents of the instant invention can be rapidly deployed in the detection of newly discovered Va and VIlla mutations.

The methods of the present invention can also be generally applied to other plasma factors in the blood clotting pathways. The clotting factors are typically activated from their inactive precursor state by proteolytic cleavage at specific sites.

Therefore, mutations at the activating cleavage site may prevent such activation, resulting in hemorrhage. Nucleic acid ligands to mutant clotting factor proteolytic activation sites could be used to detect such mutations in the presence of the proteins that normally catalyze the cleavage. Conversely, many other factors are inactivated by proteolytic cleavage by enzymes other than APC; still other factors are inhibitors of the blood clotting pathway that can only inhibit when they have undergone a proteolytic cleavage event. For example, thrombin, when bound to thrombomodulin, proteolytically cleaves and activates APC. It would be expected that mutations in APC at the site cleaved by thrombin would render APC incapable of being activated, leading to thrombosis. Nucleic acid ligands against mutations in the thrombin cleavage site of APC could therefore be used in the presence of a thrombin/ thrombomodulin complex to detect such mutations. In all cases, the crucial concept is the use of a nucleic acid ligand that either specifically recognizes the mutated cleavage site, or recognizes sites on either side of the mutated cleavage site. The nucleic acid ligands are used in an assay in combination with the proteolytic enzyme that normally cleaves at the wild-type version of the cleavage site. Nucleic acid ligands that bind to the mutated cleavage site do so without any cross-reactivity with the wild-type cleavage site in the sample, as this is all cleaved by the proteolytic enzyme. Nucleic acid ligands that bind on either side of the cleavage site also do not cross-react with wild-type protein, as this is also cleaved by the proteolytic activity of APC.

C. Detection of Binding of Nucleic Acid Ligands to Factor V Leiden I. Dot Blot Assavs In one embodiment of the invention, the nucleic acid ligands are used in a dot blot assay, well known to those skilled in the art. In this assay, the blood

sample is first treated with a proteolytic enzyme, such as thrombin, and APC as described above. A portion of the sample, or a dilutions series thereof, is spotted by a liquid dispensing-means onto a solid support. The solid supports of the present invention include, but are not limited to, membranes, charged paper, nylon, beads, or virtually any other type of solid support. Proteins in the sample become stably associated with the surface of the solid support, and the surface can then be probed with a nucleic acid ligand that is labeled with a detectable moiety. Methods for performing dot blots using bodily fluids and nucleic acid ligands are described in commonly assigned United States Patent Application Serial No. 08/628,356, filed April 5,1996, entitled"Method of Detecting a Target Compound in a Substance Using a Nucleic Acid Ligand", herein incorporated by reference. Any method known in the art for non-specifically immobilizing a protein on a solid support is contemplated.

In some embodiments, the blood sample attaches non-specifically to the solid support through covalent or non-covalent interactions with the surface of the solid support. In other embodiments, the solid support comprises antibodies to Factor Va.

The antibodies recognize sites distinct from those recognized by the nucleic acid ligands, and are used to specifically and non-covalently attach Factor Va to the solid support. In preferred embodiments, the antibodies bind to an epitope on Factor Va that is N-terminal to the mutated APC cleavage site. In this way, both wild-type Factor Va and Factor V Leiden are localized to the solid support, but only the Factor V Leiden reacts with the nucleic acid ligand that recognizes the mutated APC cleavage site.

In order to detect immobilized mutated forms of Factor V Leiden, a suitable detectable label must be attached to the nucleic acid ligand. The attachment of a suitable detection system such as an enzyme or a fluorophore to nucleic acid ligands is not problematic and in some cases (fluorophores) can be attached during the chemical synthesis of the ligand itself. The use of bioluminescent and chemiluminescent substrates allows the detection of target compound concentrations in the 10-11-10-"M range. The sensitivity of this assay can be further increased by using bioluminescence

or chemiluminescence when nucleic acid ligands are attached to alkaline phosphatase (AP).

The enzymes most commonly used in this procedure, alkaline phosphatase and horseradish peroxidase, form a colored product which can be detected by visual inspection of the membrane. The high sensitivity of this type of reagent has both advantages and disadvantages. Results are obtained quickly, but the use of an extremely sensitive detection method can be confusing, especially if the background staining level is high. If the signal-to-noise ratio is too low or the optimal amount of protein is not immobilized on the solid support, and the desired information cannot be obtained, the membrane cannot easily be reprobed or stripped. However if radiolabel is used, the time of autoradiographic exposure can be varied to obtain the optimal signal, the solid support can be reprobed easily, and with less buildup of background signal than is possible with enzyme-conjugated detection. However, the speed of detection is often an overriding concern, and the enzyme-conjugated protocol may be the method of choice.

II. Detection Involving a Solid Support Bound Nucleic Acid Ligand to Factor V Leiden In another series of embodiments, the nucleic acid ligand, rather than the blood sample, is attached to a solid support. Solid supports that are suitable for the immobilization of nucleic acids are well known in the art, as is the chemistry used for such attachment. For example, solid supports and attachment chemistries are described in detail in WO 98/20020, specifically incorporated herein by reference in its entirety. In these embodiments, the blood sample is treated with a proteolytic enzyme and APC, then contacted with the solid support-bound nucleic acid ligand.

Following incubation, the solid support is washed to remove proteins that are not bound by the nucleic acid ligand.

Binding of the Factor V Leiden protein to the nucleic acid ligand can be detected in this scenario in a number of ways. Suitable methods for detecting the binding of target to a solid support-bound nucleic acid ligand are discussed in great detail in co-pending and commonly assigned United States Patent Application Serial

No. 08/990,436, filed December 15,1997, entitled"Nucleic Acid Ligand Diagnostic Biochip"and incorporated herein by reference in its entirety.

In one embodiment, the binding is monitored using the technique of surface plasmon resonance (SPR). In this technique, the nucleic acid ligand is immobilized on a gold or silver film on the surface of a prism ; the metal film is then incubated with the blood sample that has been treated with thrombin and APC. Therefore, the metal film is at the prism-liquid interface. Light is directed through the prism towards the medium, and above a critical angle, total internal reflection of the light occurs. Above this critical angle, an evanescent wave extends into the medium by a distance that is approximately equal to the wavelength of the incident light. The evanescent wave excites free oscillating electrons, termed surface plasmons, in the metal film and causes them to resonate. Energy is absorbed from the evanescent wave by the electrons during this process, thereby reducing the intensity of the internally reflected light. The angle at which total internal reflection, and hence resonance, occurs is exquisitely sensitive to changes in the refractive index of the medium immediately adjacent to the metal film. When Factor V Leiden binds to a nucleic acid ligand on the surface of the film, the refractive index at this site changes, and the angle needed to cause resonance changes also. Thus in order to detect Factor V Leiden binding, a detector system is arranged in which the angle of incident light is varied, and the intensity of the reflected light is measured. Resonance occurs when the intensity of the reflected light is at a minimum. It is also possible with this technique to provide a solid support upon which nucleic acid ligands to different mutated APC cleavage sites in Factor Va are arrayed in a spatially discrete manner. Measuring the change in angle of incident light needed to bring about resonance at specific sites on the film in the presence of a test mixture can then yield information about where a binding reaction has occurred on the surface of the film. This in turn can reveal which particular APC resistant Factor Va mutation is present. A device for measuring SPR called BIAcore is commercially available from Pharmacia Biosensors. This technique is particularly well suited to highly automated high throughput analysis of blood samples.

In another embodiment, the binding is detected through the use of mass spectrometry, in particular through the use Matrix Assisted Laser Desorption and

Ionization Time of Flight (MALDI-TOF) mass spectrometry. Although MALDI-TOF instruments were once pure laboratory research tools, advances have recently been made in their design, making them a viable choice for routine diagnostic testing of biological samples. MALDI-TOF uses a laser beam to desorb and ionize material (in this case, bound Factor V Leiden) from a solid support that is coated with a light- absorbing matrix. The ions enter a time-of-flight tube, and become separated according to their mass as they drift through the tube. The ions exit the tube and strike an ion detector at different times according to their mass. Computer analysis of this spectrum can determine whether ionization products resulting from the desorption of Factor V Leiden are present. This analysis can be performed extremely rapidly, usually within less than a minute. As with SPR, the method can be used with solid supports that contain a plurality of nucleic acid ligands, each with affinity for a different target, and each attached to a discrete site. Instruments are now available that can scan a laser beam over discrete sites on the solid support, and detect the ionization product from each site. In this way, the Factor V Leiden mutation can be detected during a diagnostic test for a variety of other medical conditions.

A still further method for detecting the binding of Factor V Leiden is the technique known as multiphoton detection. This technique, described in WO 98/02750, incorporated herein by reference in its entirety, uses derivatizing agents that can label proteins with multiphoton-emitting radioisotopes. Such isotopes emit exactly coincident gamma and X-ray photons of characteristic energies upon decay.

The derivatizing step can take place after the solid support has been contacted with the blood sample that has been treated with thrombin and APC, and then washed extensively. A multiphoton detector is then used to measure gamma and X-ray emission on the solid support. This instrument is able to almost completely screen out background emission by detecting only coincident gamma and X-ray emissions with the energies expected from the derivatizing agent. Gamma and X-ray pulses that are not coincident, or are not of the expected energy, are removed from the detection signal by computer analysis. Indeed, background emission can be reduced to a level of 0.5 counts per week using this technique. As a result, it is possible to use incredibly small amounts of radioisotopes (less than 1 nanoCi ; which is below the

ambient level of radioactivity) for the labeling reactions. This makes the technique suitable for routine diagnostic testing. Furthermore, this technique can be used with a solid support with an array of nucleic acid ligands with specificities for a variety of different targets.

III. Solution Phase Methods for Detecting Factor V Leiden Mutation Other methods for detection do not rely on the use of solid supports to which either the nucleic acid ligand or the blood sample is immobilized. In these assays, the thrombin, APC and the nucleic acid ligand can simply be added to a single container and the binding reaction monitored with a spectrophotometer.

In one embodiment, the binding of Factor V Leiden to the nucleic acid ligand is detected using a ligand beacon. Ligand beacons are described in co-pending and commonly assigned United States Provisional Patent Application entitled "Homogeneous Detection of a Target through Nucleic Acid Ligand-Ligand Beacon Interaction", filed contemporaneously with the present application and specifically incorporated herein in its entirety. In preferred embodiments, ligand beacons are stem-loop nucleic acids that are capable of hybridizing to specific nucleic acid ligands only when the nucleic acid ligand is not bound to its target. The loop of the ligand beacon is complementary to at least part of the nucleic acid ligand. When the loop hybridizes to the nucleic acid ligand, the stem unwinds. Nucleotide positions in the stem that become separated from one another (such as the 5'and 3'termini) during unwinding are labeled with a quenching group and a fluorescent group. In the stem, these groups are adjacent, and so fluorescent signal from the fluorescent group is quenched by the quenching group. As the stem unwinds, the groups are separated, and the quenching group is no longer able to quench the fluorescent group. Hence, hybridization of the ligand beacon to its cognate nucleic acid ligand is accompanied by an increase in the fluorescence emission from the ligand beacon. The ligand beacon is unable to bind to the nucleic acid ligand when the latter is associated with its cognate target. In addition, the nucleic acid ligand is unable to bind to its target when hybridized to its ligand beacon.

Ligand beacons are used in a competition assay to detect the presence of Factor V Leiden. In this assay, the blood sample is treated with thrombin, APC, and a predetermined amount of the nucleic acid ligand that can recognize the mutated APC cleavage site in Factor V Leiden. The assay mixture is then added to a solution containing a ligand beacon; the ligand beacon solution has little or no fluorescence emission as all of the fluorescent groups are quenched. If none of the nucleic acid ligand is bound to its cognate target, then the ligand beacon can bind to this free nucleic acid ligand, thereby generating a fluorescent signal. If the nucleic acid ligand is complexed with Factor V Leiden, then the ligand beacon cannot bind, and the fluorescent signal remains quenched. Comparison of the change in fluorescence signal in this assay with that obtained when known amounts of Factor V Leiden, nucleic acid ligand, and ligand beacon are contacted, reveals whether the individual is homozygous wild-type, heterozygous for Factor V Leiden, or homozygous for Factor V Leiden.

Because the Factor V Leiden protein may be present in extremely small quantities in the blood sample, it may be necessary to use a signal amplification system. A suitable system is described in commonly assigned United States Provisional Patent Application Serial No. 60/068,135, field December 15,1997, entitled"System for Amplifying Fluorescent Signal Through Hybridization Cascade" and specifically incorporated herein by reference in its entirety. In this method, a first nucleic acid ligand binds to a target molecule and undergoes a conformational change that allows other nucleic acids to hybridize thereto. The nucleic acids that hybridize to the nucleic acid ligand also undergo a conformational change during hybridization that similarly allows other nucleic acids to hybridize thereto. This chain reaction of conformational change and hybridization will continue until one of the participating nucleic acids is depleted. In one such embodiment, all of the nucleic acids have a stem-loop structure, including the nucleic acid ligand that recognize the Factor V Leiden mutated APC cleavage site. The sequences of the nucleic acids are chosen so that the regions that form the stem in one member of the set will hybridize more stably to those regions of another member of the set than to one another. For example, the set could comprise sequences as follows wherein the letters A, B, and C signify a

unique sequence in the stem region,"/"signifies imperfect intramolecular base pairing, and"'"signifies a complementary sequence: (i) A/B, (ii) B'/C (iii) C'/A'.

Thus, if the nucleic acid ligand is A/B and these sequences become available for intermolecular base-pairing upon binding to the Factor V Leiden, then the A segment will then bind to the A'segment of C'/A', and the B segment will bind to the B' segment of B'/C. This in turn allows the C and C'portions of the newly bound nucleic acids to bind to their complementary sequences in C'/A'and B'/C respectively. These reactions occur because the formation of the intermolecular helical stem is more energetically favored than the intramolecular helix formation. This cascade of intermolecular helix formation between the three members of the set results in the formation of a multimolecular hybridization complex. The initiating event is the binding of the nucleic acid ligand to Factor V Leiden.

Each member of the set of nucleic acids can be labeled with a fluorescence group and a quenching group at positions that become separated upon dissolution of the stem region. As in the previous embodiments, this separation leads to a change in the spectral properties of the nucleic acids, and this change can be monitored to determine whether the target molecule is present. The cascading nature of the system means that a single nucleic acid ligand binding to Factor V Leiden can generate a very large signal.

In all of the foregoing embodiments, the assay can be performed in a single container. The blood sample is first treated to remove fibrin and cells, including platelets. This can be readily accomplished by centrifugation. The sample is then treated sequentially or simultaneously with thrombin, APC and the appropriate nucleic acid (s). The container is then transferred to a spectrophotometer which measures the spectral shift (s) that result from the nucleic acid ligand binding to Factor V Leiden. It will be appreciated by those skilled in the art that this assay can be easily automated using robots that add precise amounts of the reagents, incubate the containers at the appropriate temperatures, then transfer the containers to spectrophotometers. With such a system, high throughput analysis of blood samples is easily enabled.

It will also be recognized by those skilled in the art that any of the foregoing embodiments using ligand beacons can be equally-well adapted for use with solid supports. By way of example only, it is possible to immobilize a nucleic acid ligand on the solid support, and detect binding of Factor V Leiden by detecting the spectral shift that occurs upon binding in the presence of a ligand beacon.

EXAMPLES The following examples are given in order to illustrate only one embodiment of the instant invention. The Examples are not to be construed as limiting the scope of the invention; rather, the invention is defined by the Claims and the full scope of their equivalents.

Example 1 Obtaining a Nucleic Acid Ligand to Factor V Leiden A peptide comprising the Factor V Leiden site and amino acids on either side of the site is expressed in an appropriate expression system. This is done by cloning the DNA sequence that encodes the peptide into a suitable vector, then transforming that vector into a host cell capable of expressing the cloned sequence. Such procedures are well within the ability of those skilled in the art. The expressed Leiden peptide is then purified from the host cells. A peptide corresponding to the wild-type sequence of Factor V in this region is produced in an identical manner. The sequences of the peptides used are: Leiden: Arg Ser Leu Asp Arg Arg Gly Ile Gln Arg Ala Ala SEQ. ID. NO : 1 Wild-type: Arg Ser Leu Asp Arg Gln Gly Ile Gln Arg Ala Ala SEQ. ID. NO : 2 The peptides are then used in the SELEXTM process. First, the Leiden peptide is bound to magnetic beads, and then contacted with a pool of single-stranded nucleic acids that have a common fixed 5'end and a fixed 3'end sequence. The sequence between the fixed regions is randomized. After incubation, nucleic acids that have not

bound to the Leiden peptide are removed from the magnetic beads by pelleting the beads through the application of a magnetic field, then washing the beads extensively with buffer. Nucleic acids that bind the Leiden peptide are then denatured to remove them from the bound peptide on the magnetic beads, and the denatured nucleic acids are removed from the beads by aspiration. The nucleic acids are renatured, and are then contacted with magnetic beads to which the wild-type peptide is bound. Nucleic acids that do not bind to the wild-type peptide are removed by aspiration, and amplified using the Polymerase Chain Reaction with primers that specifically hybridize to the fixed 5'and 3'sequences. The amplified pool of nucleic acids is again contacted with the bead-bound Leiden peptide, and the selection cycle is repeated. After a nucleic acid pool with the desired properties (high affinity for Factor V Leiden, very low affinity for wild-type Factor V) is obtained, the pool is cloned into a bacterial vector and sequenced. Individual nucleic acids in the pool are then produced by conventional oligonucleotide synthesis techniques with a fluorescein molecule at the 5'end.

Example 2 Assav for the Presence of the Factor V Leiden Mutation in Blood Blood is withdrawn from an individual suspected of carrying the Leiden mutation, and centrifuged to remove cells, including platelets, and any fibrin clots.

The resulting plasma is then treated with activated protein C and thrombin in amounts sufficient to cleave all wild-type Factor V to Factor Va, and cleave all plasma-derived wild-type Factor Va between arginine 506 and glycine 507. The treated plasma is then spotted onto a dot blot membrane, along with similarly-treated plasma from individuals that are wild-type, homozygous for the Leiden mutation or heterozygous for the Leiden mutation. The dot blot is then contacted with fluorescein-labeled nucleic acid ligands to Factor V Leiden that are obtained as described in Example 1.

The blots are gently washed, and are then examined by a scanning fluorometer. The level of fluorescein signal in the plasma sample being tested is then compared with the signals on the blot from plasma taken from the wild-type, heterozygous, and homozygous controls. This comparison reveals the genotype of the individual being tested with respect to the Factor V Leiden mutation.