Thrombin-binding Aptamer Research Articles

Thrombin Ultrasensitive Detection Based on Chiral Supramolecular Assembly Signal-Amplified Strategy Induced by Thrombin-Binding Aptamer.

Thrombin plays a critical role in hemostasis and hemolysis. It is of high importance to develop a system toward thrombin detection with high sensitivity and high selectivity for both research and clinical diagnosis applications. In this paper, we developed a thrombin detection assay by taking advantage of the novel signal amplified strategy based on the chiral supramolecular assembly in physiological K+ background. This assay could detect thrombin as low concentration as about 2 pM and provided a highly specific selectivity among several common interferences. Furthermore, the assay can discriminate thrombin from other nonspecific analogous proteins with high selectivity and can be used to detect thrombin in diluted real human serum samples, which suggested its great potential for rapid detection of thrombin in the clinic.

Molecular dynamics simulations of aptamer-binding reveal generalized allostery in thrombin

Thrombin is an attractive target for antithrombotic therapy due to its central role in thrombosis and hemostasis as well as its role in inducing tumor growth, metastasis, and tumor invasion. The thrombin-binding DNA aptamer (TBA), is under investigation for anticoagulant drugs. Although aptamer binding experiments have been revealed various effects on thrombin’s enzymatic activities, the detailed picture of the thrombin’s allostery from TBA binding is still unclear. To investigate thrombin’s response to the aptamer-binding at the molecular level, we compare the mechanical properties and free energy landscapes of the free and aptamer-bound thrombin using microsecond-scale all-atom GPU-based molecular dynamics simulations. Our calculations on residue fluctuations and coupling illustrate the allosteric effects of aptamer-binding at the atomic level, highlighting the exosite II, 60s, γ and the sodium loops, and the alpha helix region in the light chains involved in the allosteric changes. This level of details clarifies the mechanisms of previous experimentally demonstrated phenomena, and provides a prediction of the reduced autolysis rate after aptamer-binding. The shifts in thrombin’s ensemble of conformations and free energy surfaces after aptamer-binding demonstrate that the presence of bound-aptamer restricts the conformational freedom of thrombin suggesting that conformational selection, i.e. generalized allostery, is the dominant mechanism of thrombin-aptamer binding. The profound perturbation on thrombin’s mechanical and thermodynamic properties due to the aptamer-binding, which was revealed comprehensively as a generalized allostery in this work, may be exploited in further drug discovery and development.

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (106M−1 order) with diminished binding to dsDNA (104M−1 order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×106M−1 and a 2:1 stoichiometry) than dsDNA (K=7.5×104M−1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.

Anti-HIV Activities of Intramolecular G4 and Non-G4 Oligonucleotides

New natural and chemically modified DNA aptamers that inhibit HIV-1 activity at submicromolar concentrations (presumably via preventing viral entry into target cells) are reported. The new DNA aptamers were developed based on known intramolecular G-quadruplexes (G4s) that were functionally unrelated to HIV inhibition [the thrombin-binding aptamer and the fragment of the human oncogene promoter (Bcl2)]. The majority of previously described DNA inhibitors of HIV infection adopt intermolecular structures, and thus their folding variability represents an obvious disadvantage. Intramolecular architectures refold correctly after denaturation and are generally easier to handle. However, whether the G4 topology or other factors account for the anti-HIV activity of our aptamers is unknown. The impact of chemical modification (thiophosphoryl internucleotide linkages) on aptamer activity is discussed. The exact secondary structures of the active compounds and further elucidation of their mechanisms of action hopefully will be the subjects of future studies.

Label-free monitoring of DNA polymerase activity based on a thrombin-binding aptamer G-quadruplex

We have developed a label-free assay for the detection of DNA polymerase activity based on a thrombin-binding aptamer (TBA) G-quadruplex. In the presence of DNA polymerase, the 3′-OH termini of the hairpin substrate are immediately elongated to replace the TBA, which can be recognized quickly by the ThT dye and results in an increase of fluorescence. This method is highly sensitive with a detection limit of 0.1 U/mL. It is simple and cost-effective without any requirement of labeling with a fluorophore-quencher pair. Furthermore, the proposed method can also be applied to analyze the inhibition of DNA polymerase, which clearly indicates that the proposed method can be applied for screening of potential DNA polymerase inhibitors.

The effect of l-thymidine, acyclic thymine and 8-bromoguanine on the stability of model G-quadruplex structures

BackgroundGuanine-rich oligonucleotides are capable of forming tetrahelical structures known as G-quadruplexes with interesting biological properties. We have investigated the effects of site-specific substitution in the loops and in the tetrads model G-quadruplexes using thymine glycol nucleic acid (GNA) units, l-thymidine and 8-Br-2′-deoxyguanosine. MethodsModified oligonucleotides were chemically synthesized and spectroscopic techniques were used to determine the relative stability of the modified G-quadruplex. The double 8-BrdG-modified quadruplexes were further characterized by Nuclear Magnetic Resonance. Binding to thrombin of selected quadruplex was analyzed by gel electrophoresis retention assay. ResultsThe most interesting results were found with a 8-bromoG substitution that had the larger stabilization of the quadruplex. NMR studies indicate a tight relationship between the loops and the tetrads to accommodate 8-bromoG modifications within the TBA. ConclusionsThe substitutions of loop positions with GNA T affect the TBA stability except for single modification in T7 position. Single l-thymidine substitutions produced destabilization of TBA. Larger changes on quadruplex stability are observed with the use of 8-bromoG finding a single substitution with the highest thermal stabilization found in thrombin binding aptamers modified at the guanine residues and having good affinity for thrombin. Double 8-BrdG modification in anti positions of different tetrads produce a conformational flip from syn to anti conformation of 8-Br-dG to favor loop-tetrad interaction and preserve the overall TBA stability. General significanceModified guanine-rich oligonucleotides are valuable tools for the search for G-quadruplex structures with higher thermal stability and may provide compounds with interesting protein-nucleic acid binding properties. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Backbone modified TBA analogues endowed with antiproliferative activity

BackgroundThe thrombin binding aptamer (TBA) is endowed with antiproliferative properties but its potential development is counteracted by the concomitant anticoagulant activity. MethodsFive oligonucleotides (ODNs) based on TBA sequence (GGTTGGTGTGGTTGG) and containing l-residues or both l-residues and inversion of polarity sites have been investigated by NMR and CD techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay), and their resistance in fetal bovine serum have been tested. ResultsCD and NMR data suggest that the investigated ODNs are able to form right- and left-handed G-quadruplex structures. All ODNs do not retain the anticoagulant activity characteristic of TBA but are endowed with a significant antiproliferative activity against two cancerous cell lines. Their resistance in biological environment after six days is variable, depending on the ODN. ConclusionsA comparison between results and literature data suggests that the antiproliferative activity of the TBA analogues investigated could depends on two factors: a) biological pathways and targets different from those already identified or proposed for other antiproliferative G-quadruplex aptamers, and b) the contribution of the guanine-based degradation products. General significanceModified TBA analogues containing l-residues and inversion of polarity sites lose the anticoagulant activity but gain antiproliferative properties against two cancer cell lines. This article is part of a Special Issue entitled “G-quadruplex” Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Gravimetric biosensor based on a 1.3 GHz AlN shear-mode solidly mounted resonator

We investigate the performance of solidly mounted resonators based on Ir/tilted-AlN/Ir piezoelectric stacks as biosensors. These films are deposited by varying the pressure, the cathode power and the temperature of a two-step process based on depositing (00·2)-tilted AlN active layers over an (10·3)-oriented AlN seed layer. To minimize the influence of the temperature coefficient of frequency on the stability of the biosensor, we use insulating acoustic mirrors made of layers of SiO2 and amorphous TaOx with non-λ/4 thicknesses, which enables to reduce the TCF to −14ppm/°C. The mass loading of the resonators with SiO2 thin films results in a sensitivity of 1800kHz/pg·cm2. Surface functionalization consists on the binding of silane groups on plasma oxidized SiO2 surfaces. After a glutaraldehyde link, streptavidin is bonded to the surface to receive biotinylated receptors for several species. We test thrombin-binding aptamer (TBA29 against thrombin, and IgG antibody against immunoglobulin). The sensors response to species of different molecular weight like TBA-29 (9.75kDa) or IgG antibody (150kDa) is monitored. Finally, we assess the response of the biosensors to different thrombin concentrations (ranging from 4nM to 270nM) on surfaces functionalized with the TBA29 aptamer.

Thioflavin T as a fluorescence light-up probe for both parallel and antiparallel G-quadruplexes of 29-mer thrombin binding aptamer.

A wide range of pathologies have been targeted with bimodular aptamers that contain both G-quadruplex (G4) and duplex motifs, while the structures and functions are poorly understood. G4-selective fluorescent dyes have served as facile tools to probe G4s, but not for bimodular aptamers, yet. Here, taking the 29-mer thrombin binding aptamer (TBA29) as an example, we demonstrated that 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium (ThT) was the most effective dye compared to NMM and PPIX in recognizing TBA29. Binding studies indicate that ThT recognized TBA29 via distinct buffer-dependent mechanisms. Specifically, ThT induced the formation of a bimolecular parallel G4 in cation-deficient buffer, showing 341-fold fluorescent enhancement. The competitive binding of thrombin disrupted the complex, leading to the monotonic fluorescence decrease. A similar mechanism was previously reported for the interaction between ThT and the 15-mer thrombin binding aptamer (TBA15). However, TBA29 bound with ThT in a more favorable state than TBA15, showing hyperchromic effects and two times stronger fluorescence enhancement. Differently, ThT bound with antiparallel TBA29/TBA15 in an intercalating/groove binding mode in 100mM KCl, generating 181/28-fold fluorescence enhancement, respectively. These results revealed that ThT recognized both parallel and antiparallel G4s of TBA29 more efficiently than it recognized TBA15. The duplex structure of TBA29 may play an important role in its interaction with ThT. Our study broadens the application of ThT in screening G4 to bimodular aptamers and provides some insights into the structures of TBA29, along with the interaction between ThT and TBA29. Our study also is useful for the development of structure-switching-based biosensors using bimodular aptamers. Graphical abstract The buffer-dependent binding mechanisms of ThT with TBA29, and the competitive (top)/noncompetitive (bottom) binding of thrombin with TBA29-ThT complex.

G-Quadruplex Aptamers to Human Thrombin Versus Other Direct Thrombin Inhibitors: The Focus on Mechanism of Action and Drug Efficiency as Anticoagulants.

Thrombin is a key enzyme of blood coagulation system which has multiple functions including pro- and anticoagulant, platelet aggregating and inflammatory activities. Unsurprisingly, this enzyme has been a target for anticoagulant drug development for decades. Among the most interesting direct thrombin inhibitors with intravenous administration route are the following ones: 1) hirudins, proteins with bivalent binding mode to the thrombin, 2) bivalirudin, the peptide with bivalent binding mode to the thrombin, 3) argatroban, the chemical that binds to the thrombin active site, and 4) G-quadruplex DNA aptamers, structured oligonucleotides with an affinity to protein-binding site of the thrombin. Efficiency of all these inhibitors has been studied in vivo in preclinical and clinical trials, as well as in vitro with various tests, allowing to compare them thoroughly. In the review three levels of comparison were used to highlight the features of each inhibitor: 1) thrombin inhibition constants as a characteristic of inhibitor potency in simple enzymatic system; 2) inhibition of fibrin fiber formation and thrombin generation in coagulation cascade as a characteristic of anticoagulant potency in human blood plasma; and 3) therapeutic doses used and therapeutic profiles obtained after intravenous administration into animals and humans. The data clearly demonstrate weak and strong aspects of thrombin binding aptamers providing a solid background for further novel anticoagulant development. </p><p&gt.

A Simple Molecular Rotor for Defining Nucleoside Environment within a DNA Aptamer-Protein Complex.

The simple 5-furyl-2'-deoxyuridine ((Fur)dU) nucleobase exhibits dual probing characteristics displaying emissive sensitivity to changes in microenvironment polarity and to changes in solvent rigidity due to its molecular rotor character. Here, we demonstrate its ability to define the microenvironment of the various thymidine (T) loop residues within the thrombin binding aptamer (TBA) upon antiparallel G-quadruplex (GQ) folding and thrombin binding. The emissive sensitivity of the (Fur)dU probe to microenvironment polarity provides a diagnostic handle to distinguish T bases that are solvent-exposed within the GQ structure compared with probe location in the apolar duplex. Its molecular rotor properties then provide a turn-on fluorescent switch to identify which T residues within the GQ bind specifically to the protein target (thrombin). The fluorescence sensing characteristics of (Fur)dU make it an attractive tool for mapping aptamer-protein interactions at the nucleoside level for further development of modified aptamers for a wide range of diagnostic and therapeutic applications.

An electrochemical label-free and sensitive thrombin aptasensor based on graphene oxide modified pencil graphite electrode

In this work, we tactfully constructed a novel label-free electrochemical aptasensor for rapid and facile detection of thrombin using graphene oxide (GO) and thrombin binding aptamer (TBA).The strategy relies on the preferential adsorption of single-stranded DNA (ssDNA) to GO over aptamer-target complexes. The TBA-thrombin complex formation was monitored by differential pulse voltammetry (DPV) using the guanine oxidation signal. In the absence of thrombin, the aptamers adsorbed onto the surface of GO leading to a strong background guanine oxidation signal. Conversely, in the presence of thrombin, the conformational transformation of TBA after incubating with the thrombin solution and formation of the aptamer-thrombin complexes which had weak binding ability to GO, leads to the desorption of TBA-thrombin complex from electrode surface and significant oxidation signal decrease.The selectivity of the biosensor was studied using other biological substances. The biosensor's signal was proportional to the thrombin concentration from 0.1 to 10nM with a detection limit of 0.07nM.Particularly, the proposed method could be widely applied to the aptamer-based determination of other target analytes.

A novel nanosensor composed of aptamer bio-dots and gold nanoparticles for determination of thrombin with multiple signals

Thrombin is a crucial multifunctional enzyme involved in many physiological and pathological processes. Its detection is of great importance. In this work, a novel bio-dots nanosensor for detection of thrombin with colorimetric, fluorometric and light-scattering signals is developed. This nanosensor is composed of thrombin-binding aptamer bio-dots (TBA-dots) and gold nanoparticles (AuNPs), where TBA-dots serve as fluorometric reporter and AuNPs function as multiple roles as colorimetric reporter, light scattering reporter and fluorescence quencher. TBA-dots retain inherent structures of aptamer to specifically interact with thrombin and simultaneously induce the aggregation of AuNPs. The mechanism of the sensing system is based on distance-dependent aggregation of AuNPs and fluorescence resonance energy transfer (FRET). The nanosensor needs no further surface functionalization required for the as-prepared bio-dots and AuNPs, which provides a sensitive method for the selective detection of thrombin with a detection limit as low as 0.59nM. In addition, it provides a brand new perspective for bio-dots and its potential use in bioanalysis.

A Comparative Study of G-Quadruplex Unfolding and DNA Reeling Activities of Human RECQ5 Helicase

RECQ5 is one of five members of the RecQ family of helicases in humans, which include RECQ1, Bloom (BLM), Werner (WRN), RECQ4, and RECQ5. Both WRN and BLM have been shown to resolve G-quadruplex (GQ) structures. Deficiencies in unfolding GQ are known to result in DNA breaks and genomic instability, which are prominent in Werner and Bloom syndromes. RECQ5 is significant in suppressing sister chromatid exchanges during homologous recombination but its GQ unfolding activity are not known. We performed single-molecule studies under different salt (50–150 mM KCl or NaCl) and ATP concentrations on different GQ constructs including human telomeric GQ (with different overhangs and polarities) and GQ formed by thrombin-binding aptamer to investigate this activity. These studies demonstrated a RECQ5-mediated GQ unfolding activity that was an order of magnitude weaker than BLM and WRN. On the other hand, BLM and RECQ5 demonstrated similar single-stranded DNA (ssDNA) reeling activities that were not coupled to GQ unfolding. These results demonstrate overlap in function between these RecQ helicases; however, the relatively weak GQ destabilization activity of RECQ5 compared to BLM and WRN suggests that RECQ5 is not primarily associated with GQ destabilization, but could substitute for the more efficient helicases under conditions where their activity is compromised. In addition, these results implicate a more general role for helicase-promoted ssDNA reeling activity such as removal of proteins at the replication fork, whereas the association of ssDNA reeling with GQ destabilization is more helicase-specific.

Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.

Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K+, Na+, NH4+, Ba2+, and Sr2+; on the contrary, Mn2+ was coordinated in the grooves, outside the G-quadruplex. K+ or Na+ coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K+ coordination provided the well-known high inhibitory activity of the aptamer, whereas Na+ coordination supported low activity. Although NH4+ coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba2+ and Sr2+ coordination. Mn2+ coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a different sequence of loops in the G-quadruplex module provided an equilibrium of antiparallel and parallel G-quadruplexes that shifted with cation binding. In conclusion, structures of G-quadruplex aptamers are flexible enough and are fine-tuned with different cation coordination.

A label-free electrochemical aptasensor based on graphene oxide/double-stranded DNA nanocomposite

A novel label-free electrochemical impedance aptasensor based on a gold nanoparticles/double-stranded DNA-graphene (AuNPs/dsDNA-GO) nanocomposite modified glassy carbon electrode was presented for quantitative determination of thrombin. GO was covalently functionalized with dsDNA via a facile amidation process, and then AuNPs were electrodeposited onto the surface of dsDNA-GO. The morphology, conductivity and interaction of the as-prepared nanocomposites were characterized by scanning electron microscopy, cyclic voltammetry, electrochemical impedance spectroscopy (EIS), Raman and Fourier transform infrared spectroscopy. The thrombin-binding aptamer (TBA) was conjugated to AuNPs via gold-thiol chemistry to construct electrochemical aptasensing platform, and the specific recognition between TBA and thrombin was monitored by EIS. Under optimum conditions, thrombin could be quantified in a wide range of 0.1–100nM (R2=0.9960) with low detection limit of 0.06nM (S/N=3).

An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection.

We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.

Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model.

Thrombin-binding aptamer (TBA) with the sequence 5′GGTTGGTGTGGTTGG3′ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

Different duplex/quadruplex junctions determine the properties of anti-thrombin aptamers with mixed folding.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Design of Potent and Controllable Anticoagulants Using DNA Aptamers and Nanostructures.

The regulation of thrombin activity offers an opportunity to regulate blood clotting because of the central role played by this molecule in the coagulation cascade. Thrombin-binding DNA aptamers have been used to inhibit thrombin activity. In the past, to address the low efficacy reported for these aptamers during clinical trials, multiple aptamers have been linked using DNA nanostructures. Here, we modify that strategy by linking multiple copies of various thrombin-binding aptamers using DNA weave tiles. The resulting constructs have very high anticoagulant activity in functional assays owing to their improved cooperative binding affinity to thrombin due to optimized spacing, orientation, and the high local concentration of aptamers. We also report the results of molecular dynamics simulations to gain insight into the solution conformations of the tiles. Moreover, by using DNA strand displacement, we were able to turn the coagulation cascade off and on as desired, thereby enabling significantly better control over blood coagulation.