WO2008134069A2 - Anchored ligand agents - Google Patents

Abstract

The invention provides novel therapeutic agents comprising ligand molecules such as aptamers linked to a molecular anchor (MA). In the absence of a MA, ligands rapidly diffuse into bulk solution after dissociation from a target molecule. With a MA tightly bound to the target molecule, anchored ligand molecules of the invention remain in close proximity to the active site on the target molecule, even after dissociating from the target, permitting rapid re- binding of ligand to its target. Reversibility of binding to the active site can be effectively and rapidly achieved using antidotes based on the complementary DNA sequences. A preferred embodiment in accordance with the invention is a highly efficient anticoagulant agent that demonstrates binding efficiency to thrombin that is increased many fold relative to a corresponding unanchored aptamer-based ligand.

Description

ANCHORED LIGAND AGENTS

RELATED APPLICATIONS

This application claims the benefit of US Provisional Application No.: 60/914,242, filed April 26, 2007, the entire contents of which are expressly incorporated herein by refrence.

STATEMENT OF U.S. GOVERNMENT INTEREST

Funding for the present invention was provided in part by the Government of the United States under Grant Nos.: NIH GM66137; NIH GM 079359 and NSF EF0304569. Accordingly, the Government of the United States may have certain rights in and to the invention.

FIELD OF THE INVENTION

The invention relates generally to agents including therapeutic agents and drugs that act as ligands that specifically bind to molecular targets such proteins. Binding of the agent to the target mediates a biological activity of the target molecule. In one aspect the invention relates to anticoagulant agents that act as ligands that selectively bind to proteins of the blood coagulation cascade, to inhibit their blood clotting activities.

BACKGROUND OF THE INVENTION A vast number of biological reactions are controlled by the interaction of an effector molecule, or "ligand" with a "binding site," or "active site" in or on a target molecule. Well known among such interactions are ligand-receptor interactions, antigen-antibody interactions, and interactions between nucleic acid ligands such as aptamers and protein-based target molecules. The mechanism of action of many modern drugs is to either stimulate or inhibit a target molecule (e.g., a receptor on a cell surface, or a circulating molecule such as blood coagulation factor) by specifically binding to the target molecule. Thus, the potency of the drug will depend in part on the efficiency with which the drug is able to bind to its target molecule.

Unfortunately, many potentially useful biologies that mediate important physiological effects in vivo are limited in their suitability for drug development, due to low efficiency of binding to their target molecules. There is an unmet need for innovations in drug design that could enhance the binding efficiency of potential therapeutic molecules to their targets. Overcoming this difficulty would pave the way for development of new classes of drugs with enhanced binding capacity for treatment of a wide variety of disorders.

SUMMARY OF THE INVENTION

The invention provides a new class of molecular agents having greatly increased binding efficiency for a wide range of protein-based molecular targets, such as many known receptors and other proteins that serve as targets for a wide variety of drugs in current use.

One aspect of the invention is an anchored ligand agent for efficient binding to a target protein. The agent comprises: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a target protein or peptide; a nucleic acid molecule comprising a binding portion for binding to a second site in said target protein or peptide; and a molecule comprising a linker portion that joins said anchoring nucleic acid to said binding nucleic acid.

In one preferred embodiment, the anchoring portion of the agent ("molecular anchor, MA") can bind to the first site on the target protein with a binding affinity (Kd) of about 0.7 nM.

The second site in the target protein or peptide is associated with a specific biological activity, upon binding with a ligand. An anchored ligand in accordance with the invention is designed to specifically bind to such a biologically active site in the target molecule, inorder to effect a desired biological response. In some anchored ligand agents, at least one of the nucleic acid molecules is DNA-based ligand such as an aptamer.

In other embodiments, the invention provides bivalent molecules, e.g., comprising DNA- based ligands such as an aptamers, optionally connected by a linker. Exemplary bivalent molecules are set forth in Example 2. The linker can be a molecule of sufficient length to permit simultaneous binding of the anchoring portion to the first site on the target molecule, and binding of the binding portion to the second site on the target protein, wherein said binding results in a biological response.

Anchored ligand agents in accordance with the invention can comprise hydrophilic linker molecules (typically monomers) of variable lengths, such as a polyethylene glycol (PEG), a poly vinyl alcohol (PVA), a polyglycolide, a vinyl ether, or a phosphoramidite. In another aspect, there is also provided an anticoagulant agent comprising: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a protein associated with blood coagulation; a nucleic acid molecule comprising a binding portion for binding to a second site in said blood coagulation protein; and a linker portion joining said anchoring nucleic acid to said binding nucleic acid.

In some embodiments, at least one of the nucleic acid molecules is an aptamer that specifically binds to the blood coagulation protein thrombin. Anticoagulant agents in accordance with the invention exhibit increased thrombin-inhibiting ability, relative to that of a thrombin-inhibiting aptamer that is not linked to an anchoring portion. Other aspects of the invention are discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. FIG. IA is a schematic diagram illustrating the equilibrium between binding to a target molecule 105 and dissociation from the target molecule 105 by a free ligand 115 and a competing substrate molecule 120.

FIG. IB is a schematic diagram illustrating the equilibrium between binding and dissociation from a target molecule 105 by an anchored ligand 140 which comprises one portion of an anchored ligand agent 150 in accordance with an embodiment of the invention, and a competing substrate molecule 120. Because the anchored ligand is molecularly anchored close to the target molecule it is not free to diffuse away from the target following dissociation. This proximity to the target greatly increases the probability of re-binding to the target molecule. Hence the binding efficiency of the anchored ligand agent is greatly increased, as compared with a free ligand having the same chemical structure.

FIG. 2 is a schematic diagram illustrating the design and mechanism of action of an anchored ligand agent effective as an anticoagulant agent with thrombin inhibitory activity, as compared with an unanchored ligand, in accordance with the invention. (2A): Without the molecular anchor (MA), the ligand (15Apt) diffuses away from the thrombin molecule into the bulk solution immediately after dissociation. (2B) 15 Apt is held by the MA, permitting rapid re- binding to thrombin after dissociation, greatly increasing the binding efficiency between the 15- Apt and thrombin.

FIG. 3 is a graph showing comparison of the normalized clotting times of thrombin bound to different aptamer inhibitors. Clotting time of thrombin alone was defined as 1. The inset is a graph showing comparison of clotting times for anchored ligand agents in accordance with the invention having the indicated numbers of linker molecules (spacers).

FIG. 4 is a graph depicting real time monitoring of light scattering intensity generated by the coagulation process in the presence of different aptamers or linked aptamers agents in accordance with the invention. Fibrinogen was added at 0 second. FIG. 5 is a graph showing effect of the complementary sequence (cDNA of 15Apt) on anticoagulation of A-8-MA, a thrombin inhibitor in accordance with the present invention. Scattering intensity of the thrombin, A-8-MA and fibrinogen reaction mixture was monitored. Excess cDNA of 15 Apt was added at around 500^th second.

FIG. 6A-B are schematics of the exemplary molecules of the invention, (a) 15Apt, monovalent ligand, has constant ON and OFF and diffuses into bulk solution immediately after dissociation from thrombin, resulting in low inhibitory function, (b) In contrast, when linked to

27Apt to form a bivalent ligand, 15 Apt can rapidly return to the binding site after dissociation due to confined molecular diffusion by 27Apt that is still in the bound state to thrombin.

FIG 7 depicts a comparison of the normalized clotting times of thrombin bound to different NA inhibitors. Clotting time of thrombin alone was defined as 1, and the relative values based on it are plotted. 15Apt alone showed a threefold increase of the clotting time, but any delay was observed from the 27Apt-treated sample. Among bivalent NA candidates (Bi-xS), Bi-

8S is the best inhibitor with the longest clotting time. The replacement of 27Apt by a random sequence, as in Bi'-8S, causes almost complete loss of anticoagulant function, mainly due to the lack of bivalency and interaction between the scrambled sequence and 15Apt.

FIG. 8 depicts real-time monitoring of scattering light generated by the coagulation process in the presence of different monovalent or bivalent NA ligands (Bi-xSs). After the coagulation is initiated by adding fibrinogen to each sample, the reaction kinetics varied depending on the ligands. The initial reaction rate of each sample was calculated (scattering signal increase divided by time, cps/sec) and then plotted in the inset. This result is consistent with the clotting test. As the number of spacers increased, the reaction rate went down and then up (inset). Results show that the Bi-8S is the best design of bivalent NA inhibitor.

FIG 7A-C depict a comparison of binding kinetics, (a) Cartoon to describe the Ic_0n' measurement, (b) Cartoon to describe the k_ofr' measurement, (c) Real-time fluorescence signal change of Ic_0n' measurement. After thrombin was added, each sample mixture showed fluorescence decay. The decreasing rate was comparable in both cases. According to the calculation of the initial reaction rate, Bi-8S exhibited a 1.2 times faster Ic_0n' than did 15Apt. (d) Real-time fluorescence signal change of k_ofr' measurement. Free 15 Apt MBA (green line) showed very rapid hybridization kinetics with its target DNA. Thrombin-bound 15 Apt MBA (blue) showed slower hybridization kinetics compared to the free form. Interestingly, thrombin- bound Bi-8S MBA (red) showed a 51.7 times slower dissociation rate. The k_a' of 15Apt domain of Bi-8S is about 62 times stronger than free 15Apt.

FIG. 8 depicts reversible inhibitory function. Red: T- 15 Apt was added at around 200 seconds to the incubation of Bi-8S, thrombin and fibrinogen. Black: fibrinogen was added to thrombin at 0 seconds in the absence of any inhibitors. Blue: Bi-8S incubated with thrombin and fibrinogen (no T-15Apt).

FIG. 9A-B depict comparison of anticoagulant potency of Bi-8S and 15Apt using human plasma and aPTT and PT measurements, (a) shows dosage-dependent aPTT plotted for each NA inhibitor, and the maximal aPTT is shown inside the figure, (b) shows dosage-dependent PT, and the maximal PT recorded appears inside the figure.

FIG. 10A-B depict an investigation of concentration effect of T-15Apt in binding comparison, (a) 15 Apt/thrombin complex was treated with different amounts of T- 15 Apt. (b) Ma MBl /thrombin complex was treated with different amounts of T- 15 Apt. As shown in the figure, there was no noticeable kinetics change. FIG. 11 depicts an investigation of the dissociation of T'- 15 Apt. The preincubated mixture of F-T'-15Apt and short 15Apt-Q for 30 mins was treated with 15Apt. The increased fluorescence signal of the sample was obtained from the dissociation of T'-15Apt, which is very rapid reaction. Since this dissociation between 15Apt and T'-15Apt is much faster than the association of 15Apt to thrombin, it does not interfere the measurement of k'_on. DETAILED DESCRIPTION OF THE INVENTION

The stimulus for, or inhibition of, a vast number of biological reactions is based on the interaction of an effector molecule, or "ligand" with a "binding site," or "active site" in or on a target molecule. Well known among such interactions are receptor-ligand interactions, antigen- antibody interactions, and interactions between nucleic acid ligands such as aptamers with protein-based target molecules. In the design of drugs, the purpose of which is to stimulate or inhibit a target molecule, the potency of the drug will depend in great part on the efficiency with which the drug is able to bind to its target molecule. Many potential therapeutic molecules are limited in their usefulness due to low binding efficiency to their target molecules. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention addresses an aspect of this deficiency by providing a novel molecular agent useful as a drug and for many other applications. The agent comprises a nucleic acid ligand molecule useful for effecting a biological activity, linked to a molecular anchor. Agents in accordance with the invention can be used to selectively and efficiently bind to a target molecule such as a protein (for example in order to stimulate or inhibit the biological function mediated by the target molecule).

Agents in accorance with the invention are based on a novel molecular engineering strategy that provides for the anchoring of the attachment portion of the agent to the target molecule by means of a "molecular anchor" (MA) that binds with high affinity to the target molecule. In some preferred embodiments, the MA is an aptamer. According to this design, the anchored portion of the agent is tethered to the binding portion of the agent by the linker portion, which is the equivalent of a "molecular rope."

The binding portion of the agent, upon selective binding to its binding site on the target molecule, either stimulates or inibits a biological response that is mediated by the target molecule. By virtue of its tethering to the MA through the linker, the active binding portion of the agent is restricted by in its ability to diffuse very far from the vicinity of the binding site on the molecular target. Thus, the binding portion of the agent (or "ligand portion") is available to interact repeatedly with the binding site on the molecular target. In accordance with this design, the ligand portion of an agent of the invention is able to bind to the target molecule with much higher efficiency than is possible for the corresponding unanchored ligand. As will be readily apparent to those of skill in the art, the design of the novel agents is not especially limited, and in fact is amenable to a very wide range of applications. Particularly preferred agents of the invention are those comprising ligands that selectively bind to known drug targets with high affinity, effectively enhancing the potency of many known drugs. An agent in accordance with the invention comprises an attachment portion, a binding portion (also termed a "ligand portion,") and a linker portion. Particularly preferred agents comprise nucleic acid ligands in the form of aptamers that specifically bind to selected target molecules with very high affinity.

One preferred agent in accordance with the invention comprises nucleic acid probe linked to a molecular anchor (MA). The molecular anchor (MA) comprises a binding portion that binds with high affinity to a first binding site in the target molecule. A linker molecule is attached on one end to the MA and on the other end to a nucleic acid probe. The nucleic acid probe is selected to bind specifically to an "active" binding site in or on the target molecule that is associated with biological activity. In some preferred embodiments, the binding portion of the MA that effects the biological effect is a second aptamer.

The linker between the two aptamers (also termed a "spacer") can be any suitable hydrophilic molecule that exhibits minimal nonspecific interactions with biomolecules. Suitable hydrophilic polymers can include, e.g., a poly vinyl alcohol (PVA), a polyglycolide, a polyethylene glycol and a vinyl ether. Not all suitable polymers may be generally available as DNA synthesizer reagents. Commercially available linkers that permits optimal control of the length of the linker include, e.g., spacer phosphoramidites such as Spacer Phosphoramidite 18, Spacer Phosphoramidite 9, and Spacer Phosphoramidite C3 (the listed spacers having different lengths) from Glen Research (Sterling, VA). Figures IA and IB schematically illustrate various features of the design and mechanism of action of an anchored ligand agent in accordance with the invention. Both figures illustrate a schematic target molecule 105 having two binding sites, suspended in a solution. One of the binding sites on target molecule 105 is a ligand binding site 110, which is shown unoccupied in the drawing on the left in FIG. IB. The other binding site is a molecular anchor (MA) binding site 125, further discussed infra. Figure IA schematically illustrates the typical phenomenon of competitive binding of a free ligand 115 and a competing substrate molecule 120 for a single ligand binding site 110 on a target molecule 105. In the drawing on the left, the ligand binding site 110 is shown occupied by a free ligand molecule 115, whereas in the drawing on the right, site 110 is occupied by a competing substrate molecule 120 that is also present in the solution.

As is well known to those of skill in the art, the selective binding of a ligand that is free in a solution to a corresponding binding site on a target molecule represents an equilibrium in which the ligand is either associated with (bound to) the binding site on the target molecule, or it is dissociated from the binding site, and is free to diffuse away from the target molecule into the solution at large. The rate of dissociation of a particular ligand from a target molecule is governed by the strength of the molecular interaction between the ligand and the target (known as the "binding affinity"). Binding affinity is generally expressed in terms of a dissociation constant (Kd). For example, a dissociation constant in the range of 200-500 nM is considered to reflect a relatively low binding affinity (resulting in a tendancy for the ligand to dissociate from the target molecule and diffuse away from the target into the solution). By contrast, a Kd in the subnanomolar range would be considered to be a relatively high binding affinity (resulting in more persistent and frequent binding of the ligand to the target).

When a ligand molecule dissociates from its binding site in a target molecule, the site is temporarily left unoccupied, and thus is available for binding by another ligand molecule 115, or by a competing substrate molecule 120 present in the solution that is also able to bind to the ligand binding site. Figure IA illustrates a competitive binding situation in which free ligand molecules 115 compete with competing substrate molecules 120 for binding site 110. The drawing illustrates the equilibrium that exists between binding of the ligand 115 (left drawing) and binding of the competing substrate molecule 120 (right drawing). It is readily apparent from FIG. IA that the efficiency of binding of the free ligand 115 to the binding site 110 in the target molecule 105 is adversely affected by the ability of the free ligand to diffuse into the solution once it dissociates from the target molecule 105. Furthermore, dissociation of the free ligand 115 from the ligand binding site 110 leaves the site 110 free to be occupied by a competing substrate molecule 120. In the context of interactions of drugs with their target molecules in the bodies of living subjects such as human patients, it can be readily appreciated that the effectiveness of a drug (a type of "free ligand" as the term is used herein) depends in large part upon the successful and sustained interaction of the drug with its target molecule. As discussed above, many drugs that could otherwise effect an important interaction with a target molecule in vivo are limited in their usefulness due to their poor target-binding characteristics. As discussed above, the present invention provides a novel and innovative solution to the problem of low binding efficiency of a ligand and resultant diffusion of the ligand away from its ligand-binding site on a target molecule by providing agents ("anchored ligands") that are molecularly engineered to greatly increase their binding efficiency to their particular target molecules, as compared to the corresponding "unanchored" ligand free in solution. As illustrated in FIG. IB, an anchored ligand agent 150 in accordance with the invention comprises three components: a molecular anchor (MA) (or "anchor portion") 130; a binding portion (or "anchored ligand") 140 capable of selectively binding to the ligand binding site 110 on the target molecule 105; and a linker portion 135. Referring to the right-hand side of FIG. IB, there is illustrated the specific interaction of an anchored ligand agent 150 of the invention with its target molecule 105. As can be seen in the drawing, the interaction occurs at two sites: the molecular anchor portion 130 of the anchored ligand agent 150 specifically binds to the molecular anchor binding site 125 on the target molecule 105, whereas the anchored ligand portion 140 of the agent 150 specifically binds to the ligand binding site 110 on the target molecule 105. The length of the linker portion 135 of the agent 150 is proportioned to allow flexibility and appropriate positioning of both the anchored ligand portion 140 and the MA portion 130 in their respective binding pockets in or on the target molecule 105.

The drawing on the left side of FIG. IB illustrates the situation in which the anchored ligand 140 has dissociated from the binding site 110 in the target molecule 105. If free, the ligand 140 would be able to diffuse away from the target molecule 105 without constraint, as described above and illustrated in FIG. IA. However, due to its tethering to the molecular anchor 130 by means of the linker molecule 135, the anchored ligand 140 of the agent 150 is limited in the distance it can diffuse from the target molecule 105 by the length of the linker molecule 135. As a result, proximity of the anchored ligand 140 to the target molecule 105 is maintained, even after dissociation from the binding site 110, resulting in rapid re-binding of the ligand 140 to the target molecule 105. As further described infra, as compared with a free ligand 115, it is believed that the k_on of the binding reaction of an anchored ligand 140 of an anchored ligand agent 150 in accordance with the invention is greatly enhanced, while the £_off stays mostly unchanged. This leads to a significant improvement in binding effiency of the agent 150 to the target molecule 105. The topic of binding kinetics is further described below in regard to particular embodiments of the invention that are effective anticoagulation agents.

Highly Efficient Anchored Aptamer-based Anticoagulants

In one aspect, the invention provides novel agents in accordance with the invention that can act as highly efficient anticoagulants. The corresponding antidotes to these anticoagulants are also provided, as further discussed infra.

As discussed, an existing problem with many classes of drugs relates to their low efficiency. This drawback is well illustrated in the area of anticoagulant drugs, which are used for many different medical applications. Disorders of blood clotting can lead to a variety of serious health issues. For example, many heart-related medical conditions including stroke are caused by undesired blood clots blocking the blood vessels in the brain. Depending on location, the clot formation (also known as "coagulation" or "blood clotting") can cause other serious diseases, including pulmonary embolism (in the lung) and deep-venous thrombosis (generally in the leg). Recently, blood clotting has also been linked to tumor growth and metastasis in cancer patients, and it has been demonstrated that anticoagulation therapy can prove to be beneficial to the outcome of these patients [I].

Anticoagulant drugs act by regulating proteins of the blood coagulation cascade. Although some anticoagulants have been in clinical use for decades, there is recognition that improved anticoagulants are greatly needed for safer and more effective treatments, due to the low efficiency and poor stability of presently available drugs [2]. Histrorically, thrombin has been one of the preferred molecular targets for anticoagulant therapeutics. A widely used anticoagulant is heparin, which is known to inhibit the blood coagulation protein thrombin. Heparin inhibits thrombin in an indirect manner, by enhancing the activity of the natural antithrombin [3]. Unfortunately, heparin preparations exhibit diversity in molecular size, and demonstrate nonspecific binding to plasma proteins [4]. Thus, it is difficult to determine optimum drug dosage, and this uncertainty can lead to serious complications such as bleeding. Fractionated low-molecular-weight heparins are known have a more predictable anticoagulant effect; however, these products are antithrombin-dependent and are not effective to inactivate thrombin that is bound to blood clots, fibrin, or fibrin derivatives [5, 6].

For reasons as discussed above, direct thrombin inhibitors are preferable for many clinical applications. A promising development in the field of anticoagulation therapeutics is a DNA-based anticoagulant that is an aptamer, isolated by a systematic selection process [7]. Consisting of merely 15 bases in a single strand of DNA, this aptamer (abbreviated as "15Apt") has been shown to bind to exosite 1 of the thrombin molecule and to inhibit its function in the coagulation cascade. The 15Apt aptamer has high selectivity for thrombin, and demonstrates very low to no cytotoxicity, due to its DNA nature. A further unique advantage of a DNA-based anticoagulant therapy based on aptamers is that antidotes of aptamer drugs are readily available, based on their complementary DNAs (cDNAs). If necessary, the antidotes can be administered to effectively reverse the anticoagulation activity of the aptamers by virtue of the strong and rapid DNA-DNA hybridization that occurs between the aptamers and the antidotes [8, 9]. This is especially advantageous in anticoagulation therapy where, as discussed above, an over dosage of an anticoagulant can lead to serious bleeding complications.

Despite the potential of the 15 Apt aptamer as a safer and more efficient anticoagulant, it suffers from relatively low binding affinity for thrombin, with reports showing the dissociation constant (Kd) ranging from 200 to 450 nM [7, 10, 11]. The binding between the aptamer and thrombin is so weak as to even cause difficulties in observing the complex peak in capillary lectrophoresis [1 1-13].

The invention provides in one aspect improved aptamer-based anchored ligand agents that are useful as anticoagulants. As further described in Examples below, we have designed and tested novel aptamer-based therapeutics based on 15 Apt, for improved thrombin inhibition. Instead of modifying 15 Apt itself with unpredictable results, a new anticoagulant was molecularly engineered that confines the 15 Apt anchored ligand to the proximity of the thrombin molecule by means of a molecular anchor (MA).

Table 1 provides DNA sequence information pertaining to several preferred embodiments of anticoagulant agents in accordance with the invention. Table 1. DNA sequences

As illustrated in Figure 2, 15Apt is covalently linked to the MA. The 15Apt is thus held closely around thrombin even after dissociation, resulting in a much higher probability of re- binding to thrombin. The MA has similar or better affinity for thrombin than the 15Apt but binds to a different site. Because 15 Apt and MA are unlikely to be dissociated from the two binding sites on thrombin at the same time, and also because the MA is a stronger binder, the molecular assembly improves each other's binding strength by greatly increasing Ic_0n of the binding reaction while having roughly unchanged k_ofr-

One preferred sequence for the MA is a 27-mer thrombin-binding DNA aptamer (27Apt; SEQ ID NO:3) as described [17]. This aptamer is an ideal choice as a MA in combination with an anchored ligand based on 15Apt (SEQ ID NO:2) for several reasons. First, 27Apt has a strong affinity for thrombin, with an estimated Kd of 0.7 nM. Secondly, 27Apt is known to bind to a different site on the thrombin molecule than 15Apt, (i.e., exosite 2). Furthermore, simultaneous binding on exosite 1 and 2 of thrombin is known to occur [15, 16]. Finally, 27Apt can be conveniently synthesized on a DNA synthesizer and coupled to the 15 Apt. As discussed above, hydrophilic polymers suitable for use as spacers for linking the two aptamers can include, e.g., polyethylene glycol (PEG), poly vinyl alcohol (PVA), polyglycolide, polyethylene glycol, vinyl ether. One particularly preferred spacer for a thrombin-based anticoagulant in accordance with the invention comprises 18 atoms. In one embodiment, an anchored anticoagulant agent of the invention comprises SEQ ID NO:2 and SEQ ID NO:3 linked by an 18-atom polyethylene glycol (PEG) chain (-2.1 nm). In another embodiment, SEQ ID NO:2 and SEQ ID NO:3 are linked by Spacer Phosphoramidite 18 from Glen Research.

EXAMPLES This Example describes the design and testing of a DNA anticoagulant agent in accordance with the invention that comprises a molecule functioning as a molecular anchor (MA) coupled via a linker molecule to a nucleic acid ligand in the form of an anti-thrombin aptamer. This novel assembly demonstrates significantly improved anticoagulation efficiency, as compared to the free aptamer. Accordingly, this agent and those of similar design are believed to hold great potential for treating various diseases related to blood clotting disorders. Materials and Methods:

Reagents. All reagents for buffer preparation including DNA grade water and for HPLC purification were from Fisher Scientific Company L.L.C. (Pittsburgh, PA). A buffer resembling physiological conditions was used for all coagulation tests. The buffer contained 25 mM Tris- HCl at pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, ImM CaCl₂, and 5% (V/V) glycerol. Human α-thrombin was purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Fibrinogen was obtained from Sigma-Aldrich, Inc. (St. Louis, MO).

DNA synthesis and HPLC purification. All DNA synthesis reagents were from Glen Research Corp. (Sterling, VA) including DNA bases and phosphoramidites of the PEG spacer. All DNAs shown in Table 1 were synthesized with an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). The sleeping time of PEG spacer was 900 sec to maximize the coupling efficiency. For the complete cleavage and deprotection, overnight incubation with ammonia was used. After ethanol precipitation, the precipitates were re- dissolved in 0.5 ml of 0.1 M triethylammonium acetate (TEAA, pH 7.0) for further purification with high-pressure liquid chromatography (HPLC). The HPLC was performed on a ProStar HPLC Station (Varian, Inc., Palo Alto, CA) equipped with fluorescence and a photodiode array detector. A C-18 reverse phase column (Alltech Associates Inc., Flemington, NJ, C-18, 5 μM, 250x4.6 mm) was used.

DNA sequences. All DNA sequences used in this work are listed in Table 1, supra. "S" designates a PEG spacer monomer. An extra dT base was added between the aptamer and spacer to minimize potential effect of spacer on aptamer activity.

Clotting time tests. Two hundred μL of physiological buffer was added to a disposable transparent plastic cuvette (Fisher Scientific Company L.L.C., Pittsburgh, PA). Then 1 μL of 10 μM thrombin and 1 μL of 100 μM aptamer or linked aptamers (in the case of non-linked 15 Apt and 27Apt mixture, 1 μL of 100 μM of each was added) were added and incubated for 15 minutes. Subsequently, 4 μL of 20 mg/mL fibrinogen was mixed with the solution. Samples in the cuvette were examined continuously for the formation of a gel-like substance. The time when the sample became non-fluidic was recorded. Multiple oligonucleotides in different cuvettes were tested in parallel. A reaction mixture containing only thrombin and fibrinogen was always tested together with other samples as an internal standard. All clotting times were compared to, and normalized relative to the internal standard.

Light scattering tests. Reaction mixtures were prepared in the same way as in the clotting tests, with the same amounts and concentrations of samples, except that the reaction took place in a 100 μL quartz fluorescence cuvette (Starna Cells, Inc., Atascadero, CA) and the scattering light was monitored on a Fluorolog-3 spectrofluorometer (Jobin Yvon Inc., Edison, NJ). For scattering monitoring, the excitation and emission wavelengths were both set at 580 nm and the emission was detected at the right angle relative to the light excitation. In this way, the measured emission represented the scattered excitation light. This relatively longer wavelength was chosen to minimize any possible effects that the shorter wavelength light could pose on the biomolecules in the system, even though the absolute scattering intensity might be lower. The bandpass for both excitation and emission was fixed at 2 nm, which gave similar background scattering for all samples. The initial rate of scattering increase represented the relative thrombin-inhibition strength of the tested sample. Initial rates were calculated from the linear range of the early slopes of the scattering profiles.

Results: We designed MA-linked 15 Apt derivatives (A-L-MA) with various linker lengths, containing 4, 6, 8, or 10 spacers to give a length range of 8.4-21.0 nm (Table 1). Optimization of the linker length was also intended for maximizing the anticoagulant efficiency.

A typical clotting test was used to evaluate linker length effect on thrombin inhibition. Ten fold free aptamer or A-L-MA was incubated with thrombin for 15 min before excess thrombin substrate, fibrinogen, was introduced. Cleaved by thrombin, fibrinogen generates fibrin monomers that rapidly crosslink with each other to form a polymer network. Thus, the reaction solution turns from a fluidic form to a non-fluidic one, accompanied by a gel-like appearance. We recorded the time of this transition for each sample, normalized the data and compared them among the tested inhibitors. As shown in FIG. 3, 15Apt alone delayed the coagulation time by nearly 3 fold. If the same amount of each of the non-linked 15 Apt and the 27Apt was mixed and used, the coagulation time was only slightly increased (FIG. 3).

Figure 3 further shows that free 27 Apt indeed did not inhibit thrombin or noticeably affect the function of 15 Apt.

By contrast, A-L-MA with a linker of 8 spacers (A-8-MA) led to a 9 fold increase of transition time over 15Apt (27 times total over thrombin alone). Lacking thrombin inhibition capability, 27Apt worked as an MA to tightly hold 15 Apt close to thrombin to ensure that 15 Apt competed favorably for thrombin over fibrinogen. When 15 Apt was linked to a scrambled DNA by 8 spacers, the resulting A-8-random DNA showed very limited thrombin inhibition. By this demonstration, we have successfully shown that linking a non-functional binder (e.g., 27Apt MA) to a functional inhibitor (e.g., 15Apt anchored ligand) can greatly enhance the potency of the inhibitor (e.g., 15Apt). Those of skill in the art will recognize that this specific embodiment is for illustrative purposes only and that the inventive concept is applicable to a broad range of anchored ligand agents based on aptamers.

The effect of linker length is shown in the inset of FIG.3. Interestingly, the A-L-MA with 4 spacers (A-4-MA) displayed much weaker anticoagulation than other A-L-Mas, and even free 15 Apt. Without intending to be bound by theory, it is believed that this observation can be explained by the role of molecular anchors. The consequence of a linker with insufficient length is that the MA will have limited flexibility for binding and thus will prevent the anchored ligand portion of the agent from reaching the binding pocket of thrombin, leading to a greatly reduced anticoagulation capability. Six spacers seemed to be closer to the optimum length and 8 spacers gave the best inhibition of thrombin. With even longer linker length, the larger size might affect binding and intramolecular interweave could be possible, reducing the activity of 15 Apt. Considering that the diameter of thrombin is about 3-4 nm, it was somewhat unexpected to see that a linker as long as 16 nm was needed for best inhibition. It is possible that for both aptamers to take the optimal 3-dimensional position and orientation for binding, some extra linker length is required. Also, additional flexibility is likely needed to avoid too much thrombin-linker contact, and hence intermolecular repelling.

One observation not reflected by the transition time measurements was that the final coagulation product with A-8-MA or A-6-MA was much less viscous and had a more fluidic quality than those with other aptamers. To quantify this difference and obtain real-time kinetics of coagulation, we designed a more quantitative measurement based on the fact that gel formation will increase the turbidity of the reaction mixture and generate stronger scattering light. Light scattering changes during coagulation were monitored on a spectrofluorometer, and the results are shown in FIG. 4. The background scattering of the thrombin-inhibitor mixture was stable until the addition of fibrinogen. The initial rates of scattering increase reflected the relative inhibition strength of the inhibitors, with higher rates representing weaker inhibition. The trend of the anticoagulation potency correlates well with the clotting tests, while the initial rate for A-8-MA was nearly 30 times slower than that of the free 15Apt, indicating a great improvement of the anti-thrombin efficacy. We believe this enhancement is a better quantitative representation of the real situation than the clotting tests, and has substantial medical significance in achieving safer and more efficient anticoagulation therapy, lower drug dosage, and consequently fewer side effects. In addition, the total scattering increases for A-8-MA and A-6-MA were clearly lower than with other aptamers, which correlated well with the observation in clotting tests and reflected a capability of strong anticoagulants to form less dense and harmful blood clots.

Antidote effect of the cDNA of 15 Apt. To test the reversibility of this new DNA anticoagulant, excess cDNA of 15 Apt was added to the A-8-MA reaction mixture. Thrombin, A-8-MA, and fibrinogen were reacted and the light scattering was monitored as described above. About 500 seconds after fibrinogen was added to the reaction mixture, cDNA of 15Apt was added to reach a final concentration about 5 times of that of A-8-MA. An instant scattering increase was observed as shown in FIG. 5. More particularly, FIG. 5 shows the effect of the cDNA of 15Apt on anticoagulation of A-8-MA. Scattering light of the thrombin, A-8-MA and fibrinogen reaction mixture was monitored. Excess cDNA of 15 Apt was added at around 500' second. The instant increase in light scattering that was observed (FIG. 5) signals a fast and effective inactivation of A-8-MA. Therefore, introduction of the MA does not affect the reversibility of aptamer drugs.

Example 2: Bi-Balent Thrombin Molecules

Materials and Methods

Chemicals and Reagents: All DNA synthesis reagents, including 6-Fluorescein phosphoramidite, 5'-Dabcyl phosphoramidite, spacer phosphoramidite 18 and D- Deoxyphosphoramidite, were purchased from Glen Research. All reagents for buffer preparation and HPLC purification were from Fisher Scientific Company L.L.C. (Pittsburgh, PA). A buffer resembling physiological conditions was used for the buffer experiment and contained 25 mM Tris-HCl at pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, ImM CaCl₂, and 5% (V/V) glycerol. Human α-thrombin was purchased from Haematologic Technologies, Inc. (Essex Junction, VT). Fibrinogen and the sulfated hirudin fragment were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). Universal Coagulation Reference Plasma (UCRP) and thromboplastin-DL for human sample testing were purchased from Pacific Hemostasis (Cape Town, South Africa). The aPTT assay reagent was from Trinity Biotech USA (Berkeley Heights, NJ). The bivalirudin was obtained from The Medicines Company (Parsippany, NJ).

Synthesis and purification of mono- and bivalent NA ligands and their targets: To optimize the design of bivalent NA ligand, multiple candidates were designed and prepared shown in Table 2. All of them were synthesized using an ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) at lμmol scale with the standard synthesis protocol. After the complete cleavage and deprotection and ethanol precipitation, the precipitates were then dissolved in 0.5ml of 0.1 M triethylammonium acetate (TEAA, pH7.0) for purification with high-performance liquid chromatography (HPLC). The HPLC was performed on a ProStar HPLC Station (Varian Medical Systems, Palo Alto, CA) equipped with a fluorescence detector and a photodiode array detector. A C-18 reverse phase column (Alltech, Cl 8, 5μM, 250x4.6mm) was used.

Table 2. DNA sequences. S means one unit of spacer phosphoramidite. Dabcyl is a quencher and FAM is a fluorophore.

Clotting time tests: To evaluate the inhibitory potency of each NA ligand, we measured the clotting timeof each sample containing only thrombin, each nucleic acid ligand, and fibrinogen substrate in physiological buffer. The theory behind the experiment is that the mixture of sample becomes non-fluidic when the fibrinogen is digested by thrombin. As a result, the different time points of this transition can be used as an indicator. Briefly, lμL of 10 μM thrombin and 1 μL of 100 μM monovalent or bivalent nucleic acid ligand were added to a disposable transparent plastic cuvette (Fisher Scientific Company L.L.C., Pittsburgh, PA) containing 200μL physiological buffer and then incubated for 15 minutes. In the case of non-linked 15Apt and 27Apt mixture, 1 μL of 100 μM of each probe was applied. Following that, 4 μL of 20 mg/mL fibrinogen was added, and samples in the cuvette were carefully examined by tilting the cuvette to record the time when the sample becomes non-fluidic. Each experiment was performed in tandem. A reaction mixture containing only thrombin and fibrinogen was always tested together with other samples as an internal standard. All clotting times were normalized based on the internal standard and compared to it.

Real-time monitoring of the clotting reaction: To monitor the clotting event in real time,, we utilized scattering light. When fibrinogen is digested by thrombin, the mixture of sample not only becomes non-fluidic, but also cloudy. Turbidity of samples can be measured using either absorption or scattering light. To monitor the clot formation, we chose scattering light. Briefly, reaction mixtures were prepared in the same way as the clotting tests described above, except that the reaction took place in a 100 μL quartz fluorescence cuvette (Starna Cells, Inc., Atascadero, CA), and the scattering light was monitored on a Fluorolog-3 spectrofluorometer (Jobin Yvon, Inc., Edison, NJ). For scattering monitoring, the excitation and emission wavelengths were both set at 580 nm, and the emission was detected at the right angle relative to the light excitation so that the excitation light did not interfere with the light scattering signal. The initial rate of scattering increase represented the relative thrombin-inhibition strength of the tested sample. Initial rates were calculated from the linear range of the early slopes of the scattering profiles. Monitoring of the apparent k_on and k_ojf. Monitoring the binding kinetics was accomplished by modifying each inhibitor with fluorescenin and dabcyl. The sequence of each probe is shown in Table 2. To compare the &„„• of each inhibitor with thrombin, we pre-incubated 100 nM of each inhibitor with 100 nM of T'- 15 Apt. Then, the fluorescence decay, after 5 times excess of thrombin (50OnM) was added, was monitored on a Fluorolog-3 spectrofluorometer. Obtained results were used to calculate the kinetic parameters in the following way:

{F (at each time point) " F (MBA/thrombin)} / {F (MBA/T'-15Apt) — F (MBA/thrombin)}

To monitor the k_off of each inhibitor, 50OnM of T- 15 Apt was added to the pre-incubated mixture of thrombin (50OnM) and each ligand (10OnM). To generate the values of It₀/, the following equation was applied:

{F (at each time point) - F (free MBA)} / {F (MBA/T'-15Apt) — F (f_ree MBA)}

Reversible binding reaction using target DNAs: To test the reversible binding of Bi-8S, we treated the sample mixture with target DNAs of 27Apt or 15 Apt. The mixture of sample, including fibrinogen, was prepared in the same way as indicated for the clotting time. About 500 seconds after fibrinogen was added to the reaction mixture, each target DNA of either 15 Apt or

27Apt was added to reach a final concentration of about 5 times that of Bi-8S.

Human plasma tests: To evaluate the feasibility of the bivalent nucleic acid ligand as a potential anticoagulant reagent, we determined aPTT and PT for each ligand using human plasma samples. Procedures applied were those recommended by the manufacturer. For aPTT determination, 50μL of UCRP was pre-incubated at 37°C with a different amount of each ligand for 2 minutes; then 50μL of aPPT-L was added and incubated for another 200 seconds. Next, 50μL of pre-warmed CaC12 was added to initiate the intrinsic clotting cascade. Finally, the scattering signal was monitored until the signal was saturated. For PT determination, 50μL of UCRP was pre-incubated at 37°C with a different amount of each ligand for 2 minutes; then 50μL of thromboplastin-L was added to initiate the extrinsic clotting cascade. Finally, the scattering signal was monitored until the signal was saturated. For the calculation of aPTT and PT, the end time was determined to be the point where scattering signal reached half maximum between lowest and maximum points. It was repeated twice, and each set of experiments was done with one batch of plasma.

Results Thrombin aptamers and their properties. Thrombin is a multifunctional protease involved in the regulation of homeostasis. As an initiator of blood clot formation, thrombin hydrolyzes fibrinogen, and activates platelets and some blood coagulation factors that have procoagulant activity. Disorders in blood clotting are tightly linked to many serious health issues including heart attack and stroke. Therefore, thrombin is typically the target in anticoagulation therapy for these diseases. However, anticoagulant drugs currently on the market often suffer from indirect inhibition and sub-optimum selectivity, which could lead to side effects including bleeding (17-18).

There are two known thrombin NA aptamers. One is 15 base long ( 15 Apt) and binds to exosite 1, while the other, called 27Apt, is 27 base long and interacts with exosite 2 (19, 20) as shown in Figure 6. The dissociation constant K_d of 15Apt tends to be very high (up to 450 nM), depending on measurement methods(21-23), and K_d of 27Apt is approximately 0.7 nM (20) As a potential anticoagulant, only 15 Apt should have the enzymatic inhibitory functions required for thrombin-mediated coagulation since it occupies the fibrinogen-binding exosite 1. However, efforts to explore the anticoagulant effect of this aptamer have shown only limited progress due to the lack of sufficient binding strength to the exosite 1 on the target protein. Modifications on the existing aptamer itself have also been explored in order to generate an enhanced functional

NA ligand of thrombin. However, this type of construct is likely to be even more unpredictable, as its unique conformational structure can be disrupted, resulting in the loss of its binding property (24). Therefore, instead of modifying the aptamer sequence itself, we have linearly assembled two existing NA aptamers of thrombin to form a molecular assembly of thrombin aptamers. This assembly specifically improves the inhibitory function of thrombin due to the multivalent interaction mentioned above.

The design of bivalent NA inhibitor: We hypothesized that linear molecular assembly of two monovalent NA aptamers would result in a superior functional NA inhibitor of enzymatic reactions with multivalent binding properties. However, first we need to find out whether we could achieve enhanced inhibition even in the absence of multivalent interactions by simply mixing these two aptamers without covalent linkage. The second important question would be whether these two aptamers could interact with each other and subsequently cause the loss of inhibition after assembly. To address these issues, we performed typical clotting test using no aptamer, 15Apt alone, 27Apt alone, and a mixture of 15Apt and 27Apt. As shown in Figure 7, 15Apt alone delayed the coagulation time by nearly threefold, while 27Apt had no significant observed inhibition. This result was expected since 27Apt does not target the critical exosite 1. The non-linked 15Apt/27Apt mixture showed an inhibitory effect similar to the 15 Apt alone. These findings indicate that 15 Apt and 27 Apt are independent of each other and thus do not promote or interfere with each other's activity.

Next, several candidate bivalent NA ligands were designed and evaluated with the purpose of optimizing the distance between the two different NA aptamers. This step is particularly critical in designing bivalent ligands, and we initially assumed that a shorter distance between the two aptamers would result in disruption of their simultaneous binding and, hence, less effective binding and inhibition. Therefore, we designed several potential bivalent NA ligands with linkers of different lengths composed of 4, 6, 8, or 10 spacer phosphoramidites and designated to Bi-xSs, as shown in Table 2. Considering that one spacer is about 2.1 nm long and that the inner diameter of thrombin is several nanometers, this represents a sufficiently ample range of lengths; i.e., from 8.4 to 21.0 nm. Then, a clotting test was carried out to first evaluate the effect of linker length on thrombin inhibition; the results were then compared with the clotting times of the monovalent aptamer. In this test, the thrombin converts soluble fibrinogen into insoluble strands of fibrin, resulting in increasing turbidity and decreasing fluidity. The recorded time when it became completely non-fiuidic was normalized and compared among the series of potential bivalent ligands. The summary is shown in Figure 7. Briefly, as the number of spacers increased, the inhibition activity first increased and then maximized with 8 spacers. After that, a decline of inhibition was seen. Interestingly, the Bi-4S displayed a worse anticoagulation efficiency than free 15Apt. Competition between the two aptamers readily explains this phenomenon. In other words, if the distance between 15Apt and 27Apt is not sufficiently long, the binding of 27Apt will surpass that of 15Apt due to its stronger affinity to thrombin. This, in turn, will prevent 15Apt from reaching the binding pocket of thrombin and lead to a drastically reduced anticoagulation capability. However, with a sufficient linker length, Bi-8S offered a nine fold better thrombin inhibition than 15Apt alone. Contrary to our initial assumption, and considering that the size of thrombin is about 3-4 run in diameter, it was unexpected to see that a linker as long as ~16 nm (8 spacers) was needed for the best inhibition. Based on these findings, we therefore hypothesized that some extra linker length was required in order for both aptamers to wrap around thrombin and take the optimum 3 -dimensional position and orientation to interact with thrombin probably in order for minimizing interference originating from thrombin/linker contact. In spite of these results, an even longer linker length, such as that offered in Bi-IOS, was not as efficient as a shorter linker, and gave a decreased clotting time. Taken together, these results support the fact that simultaneous binding through bivalent interaction does take place and does result in improved inhibitory effect. Additional evidence was obtained from the clotting test using Bi'-8S in which 15 Apt was linked to a scrambled DNA sequence with 8 spacers. The result showed very limited thrombin inhibition. Therefore, the nine fold increase of clotting inhibition by Bi-8S over 15Apt alone is a direct result of bivalent interaction realized by molecular assembly, implying, in turn, that aptamers are ideal for the design of multivalent ligands through molecular assembly.

Monitoring inhibitory functions using light scattering: To obtain real-time kinetics of coagulation, we designed a more quantitative measurement based on the fact that gel formation increases the turbidity of the reaction mixture and generates stronger scattering light. Light scattering changes during coagulation were monitored on a spectrofluorometer, and the results are shown in Figure 8. The background scattering of the thrombin-inhibitor mixture was stable until the addition of fibrinogen. Increase of the scattering light reflected the net rate of the coagulation reaction, as this was the direct result of fibrinogen cleavage. The relative inhibition strengths of the monovalent and bivalent NA ligands were estimated from the initial reaction rates with higher rates representing weaker inhibition and the reverse for lower rates. As expected, Bi-4S produced an initial rate of 2903 cps/sec calculated from the early slope of the scattering profile, 2.8 times faster than that of 15Apt alone, 1049cps/sec, which means that 27Apt interferes with 15Apt in the binding process. In contrast, the initial reaction rates of Bi-8S and Bi-6S were much slower than the others, at 63 and 97 cps/sec, respectively. Since Bi-8S demonstrates an initial rate close to 16.6 times slower than free 15 Apt, it also represents a considerable improvement of antithrombin efficacy. Thus, the anticoagulation trend among the tested inhibitors correlates very well with the results from the clotting tests. As shown in the clotting test, Bi-IOS did not function as well as Bi-8S, resulting in an increased initial reaction rate. Bi'-8S whose sequence of 27Apt domain is replaced by the scrambled one also showed no improvement in inhibiting the clotting process. Therefore, based on the evidence gathered from both clotting test and turbidity measurement using scattering light, we conclude that Bi-8S is the best design for improved thrombin inhibition.

Binding kinetics studies: Because bivalent interaction of the aptamer assembly with thrombin increases overall binding affinity, it is proposed as the mechanism for the enhanced inhibition. Since the binding affinity is directly related to kinetic parameters, such as k_on and &_Ofr, of the thrombin/inhibitor interaction, we carried out experiments to investigate the impact of the molecular assembling on k_on and &_orr of the reaction in order to reveal what actually causes the improved inhibition. One important feature of aptamers is their binding to the target is often accompanied by changes in tertiary structures. This allows researchers to build various signal transduction mechanisms, such as FRET, into aptamers for sensitive target detection. In fact,

15Apt was among the first aptamers to be built into molecular beacon aptamers (MBAs) for protein detection based on FRET. Here, we labeled 15Apt and the 15 Apt domain of Bi-8S with a fluorophore and quencher pair to forml5Apt MBA and Bi-8S MBA 1 shown in Table 2. The compact structure of the 15 Apt bound to thrombin was expected to differ considerably from the random coil structure in solution, thus giving different fluorescence intensity. We then used these modified aptamers to study the kinetics of interactions with thrombin under different conditions.

To compare k_Off of the 15 Apt MBA and 15 Apt domain of Bi-8S MBA 1 , the full complementary target DNA of 15 Apt (T-15 Apt) was used to make the 15 Apt, when released from thrombin, inactive by forming a duplex with it (Figure 9b). At the same time, opening of the MBA should give intensive fluorescence. 10OnM of each MBA probe and 50OnM of thrombin were pre-incubated for half an hour to complete the binding reaction. Then, T-15 Apt was added to the mixture while the fluorescence signal was monitored. To normalize the fluorescence signal, MBAs fully opened by T- 15 Apt were used as the reference. One might question whether T- 15 Apt could induce the release of 15 Apt from the binding pocket. Our investigation of concentration effects of T- 15 Apt (Figure 12) shows that this was not the case. In our tests, regardless of the concentration of T- 15 Apt, the reaction kinetics was consistent, which means that T-15 Apt does not induce the release, but rather captures the released 15 Apt as a separate step. Finally, the initial rate of each reaction was calculated using the linear part of the slope. Even though values calculated this way are not the absolute k_og-' rates, they can still be useful for comparing kinetic parameters among different thrombin ligands. Determination of k_on ' for each MBA was done in a similar way (Figure 9a). Competition between thrombin and a short target DNA of 15Apt, called T'-15Apt, was studied. Briefly, each MBA was incubated with T'- 15 Apt, and then thrombin was added while the fluorescence signal was monitored. Due to the weak binding affinity between T'-15Apt and 15Apt, thrombin would compete with T'-15Apt for binding to 15Apt or the 15Apt domain of the bivalent ligands. This study was based on three assumptions: 1) that T'-15Apt only interacted with 15Apt, 2) that the binding affinity of the T'- 15Apt is identical for both MBAs, and 3) that binding of 15Apt to thrombin is more favorable than to T'-15Apt. Optimization of the T'-15Apt sequence revealed that 10 complementary bases gave the best results in terms of binding to 15 Apt and detectability of the kinetic parameters. The reason that we concluded that T'-15Apt works the best is the disassociation between 15Apt and T'- 15 Apt is not the rate limiting step but the association between 15Apt and thrombin target so that what we observed is the association rate between 15Apt and thrombin (Figure 13). The experiment result showed that such dissociation was very fast (few tens seconds to the competition). Specifically, 10OnM of each MBA and the 10-base long T'-15Apt were pre- incubated to complete the hybridization. Then, 5 times excess of thrombin was added to the mixture while the fluorescence was monitored. Immediately following that, there was sharp fluorescence signal decay. The obtained plot was normalized, and the data points for the first 100 sec were used to calculate relative reaction rates.

The relative k_ojf values obtained for monovalent and bivalent 15Apts were 1.5 and 0.029

%/sec, respectively (Figure 9d) (all measured by percentage of changes in fluorescence intensity). This means that k_of' of monovalent 15 Apt is 51.7 times faster than that of bivalent 15Apt. 15Apt MBA and Bi-8S MBA 1 have relative k_on values of approximately negative

0.00424 and 0.00498 %/sec, respectively (Figure 9c). In other words, the k_on ' of bivalent 15Apt is about similar to that of monovalent 15 Apt. Finally, we obtained the relative K_a ' values by dividing Ic_0n ' by k_off', which revealed that binding affinity of bivalent 15Apt to thrombin is about 51.7 or more times higher than that of monovalent 15 Apt. These results agree with the binding properties of other reported multivalent ligands. It is believed that, while multivalent interaction does not affect k_on ' significantly, it does alter k_off' considerably. By using the FRET strategy, we were able to measure the changes in kinetics of a single domain rather than the whole molecule, observing about 50 times higher binding affinity for the 15Apt domain. This confirms that the increased thrombin inhibition potency of the aptamer assembly originated from the kinetic changes caused by cooperative binding. As a byproduct of the study, we have also demonstrated that aptamer-based multivalent ligands make the study of kinetics using FRET quite convenient compared to antibody or small molecule based ligands.

The antidote effect of the binding aptamer. One of the unique properties of NA aptamers is that the binding can be readily regulated using the complementary sequences. Strong binding affinity of a target is critical, but reversibility of binding is equally, if not more, important. Reversibility of binding directly impacts the pharmacology of drug treatments in the sense that the side effects of drugs could then be quenched by antidotes. Based on their binding affinity as measured by the Watson-Crick base paring of complementary sequences, NA ligands and their complementary NAs are shown to be the most effective drug/antidote pairs. To demonstrate this, the antidote effects of two aptamers' target sequences, T- 15 Apt and T-27Apt, were investigated. The clotting mixture, including thrombin and fibrinogen with Bi-8S, was treated with excess T- 15 Apt and T-27Apt separately while the scattering was monitored (Figure 10). With the treatment of T- 15 Apt, immediate scattering increase was seen, and the extent of final scattering intensity was comparable to that observed without any thrombin inhibitors (Figure 10), indicating that the activity of thrombin is readily recovered by inactivation of 15 Apt, and the response is rapid. On the contrary, the clotting mixture treated with target DNA of 27Apt showed slower change in the scattering signal (data not shown), suggesting that its effectiveness in reversing the inhibition of thrombin is limited. We later used molecular beacon assay to confirm that the hybridization of 27Apt to its target was much slower than that of 15Apt and its target. It is clear that the secondary structure of 27Apt is quite stable, leading to a slower duplex formation. The second reason is that 15Apt domain was still active even when 27Apt was hybridized to its target. In conclusion, the target DNA of 15 Apt is an effective antidote, even for aptamer assembly-based therapy.

Antithrombin potency ofBi-8S: Recent studies related to its biological functions find that thrombin is critical in both blood clotting disorders and influencing tumor angiogenesis(24). Thus, after we demonstrated that Bi-8S is the best inhibitor of thrombin in buffer system, we tested Bi-8S in human plasma samples to further demonstrate the potency of the anticoagulant. Standard activated partial thromboplastin time (aPTT) (25) and Prothrombin Time (PT) (26) tests were utilized as described in Materials and Methods. The aPTT and PT are performance indicators measuring the efficacy of the contact activation pathway and extrinsic pathway of coagulation, respectively, as well as the common coagulation pathways. In each test, a different amount of each inhibitor was treated, and the obtained results were plotted using sigmoid fit. The dosage dependence is shown in Figure 11. The enhancements in delaying the coagulation triggered by both contact activation pathway and extrinsic pathway were consistently observed. The results show that the plasma samples treated with Bi-8S showed approximately five to six times longer PT and aPTT than those without any treatments, while 15Apt alone was only able to delay two to three times longer. Even though the enhancement obtained using human plasma samples was not as great as the one in a buffer system, it proved that the bivalent NA ligand can still function well in human biological fluid and give enhanced anticoagulation efficacy.

Conclusion:

In summary, by assembling two thrombin-binding aptamers with optimized linker and linker length, we have developed a NA-based high-performance bivalent ligand, which can be applied as an anticoagulant. This new design has the combined strengths of both ligands and has achieved enhanced thrombin inhibition capability, indicating its potential in biomedical applications for treating various diseases related to blood clotting disorders. Moreover, the molecular assembly approach offers a simple and noninvasive way to accomplish high performance with known protein inhibitors.

REFERENCES In some instances, the foregoing text includes numbered citations. The numbers correspond to the references shown below. It is believed that a review of the following references will increase appreciation of the present invention.

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INCORPORATION BY REFERENCE The contents of all patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.

EQUIVALENTS

Whereas methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described above. The particular embodiments discussed are illustrative only and not intended to be limiting.

Claims

What is claimed is:

1. An anchored ligand agent for efficient binding to a target protein, comprising: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a target protein or peptide; a nucleic acid molecule comprising a binding portion for binding to a second site in said target protein or peptide; and a linker portion comprising a molecule that joins said anchoring nucleic acid to said binding nucleic acid.

2. The anchored ligand agent of claim 1, wherein the anchoring portion of said agent can bind to said first site on the target protein with a binding affinity (Kd) of 0.7 nM.

3. The anchored ligand agent of claim 1 , wherein said second site in the target protein or peptide is associated with a specific biological activity, upon binding with a ligand.

4. The anchored ligand agent of claim 1 , wherein at least one of the nucleic acid molecules is an aptamer.

5. The anchored ligand agent of claim 1, wherein the linker molecule is of sufficient length to permit simultaneous binding of said anchoring portion to said first site, and of said binding portion to said second site on the target protein.

6. The anchored ligand agent of claim 1, wherein said linker molecule comprises at least one hydrophilic molecule selected from the group consisting of a polyethylene glycol (PEG), a poly vinyl alcohol (PVA), a polyglycolide, a vinyl ether, or a phosphoramidite.

7. The anchored ligand agent of claim 1, wherein the ability of the agent to effect a specific biological activity upon binding of the anchored nucleic acid ligand to said target protein is increased, relative to the ability of a corresponding unlinked nucleic acid.

8. An anticoagulant agent comprising: a nucleic acid molecule comprising an anchoring portion for binding to a first site in a protein associated with blood coagulation; a nucleic acid molecule comprising a binding portion for binding to a second site in said blood coagulation protein; and a linker portion joining said anchoring nucleic acid to said binding nucleic acid.

9. The anticoagulant agent of claim 8, wherein at least one of the nucleic acid molecules is an aptamer that specifically binds to said blood coagulation protein.

10. The anticoagulant agent of claim 8, wherein the blood coagulation protein is thrombin.

11. The anticoagulant agent of claim 10, wherein at least one nucleic acid is an aptamer that specifically binds to an exosite in the thrombin protein.

12. The anticoagulant agent of claim 11 , comprising SEQ ID NO: 1.

13. The anticoagulant agent of claim 11, comprising SEQ ID NO: 3.

14. The anticoagulant agent of claim 11 , comprising SEQ ID NO: 1 and SEQ ID NO:3.

15. The anticoagulant agent of claim 7, wherein the linker molecule comprises at least one hydrophilic molecule selected from the group consisting of a polyethylene glycol

(PEG), a poly vinyl alcohol (PVA), a polyglycolide, a vinyl ether, and a phosphoramidite.

16. The anticoagulant agent of any one of claims 10-13, wherein the number of linker molecules ranges from about 1 to about 25.

17. The anticoagulant agent of claim 10, wherein the thrombin-inhibiting ability of said agent is increased, relative to the ability of a thrombin-inhibiting aptamer that is not linked to an anchoring portion.

18. An antidote to an anticoagulant agent according to any one of claims 12-17, comprising the nucleic acid sequence represented by SEQ ID NO:2.