Gquadruplex was critical for inhibitory activity as substitution of any of the guanines reduced the inhibition of thrombin 6. The thrombin binding aptamer tba is a well characterized chairlike, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. Surprisingly, a substitution in the tgt loop induces formation of two distinct g quadruplex units in a novel dimeric structure. To record and measure the binding of thrombin to gquadruplex chips, a sandwich assay was employed whereby a secondary, cy3labelled gquadruplex aptamer, referred to as tba2 5. After an introduction to aptamers, aptamer targets, and their general uses, it discusses different applications with particular attention to the comparison between aptamerbased biosensors and methods versus the.
Loop residues of thrombinbinding dna aptamer impact g. The gquadruplex architecture is a peculiar structure adopted by guaninerich oligonucleotidic sequences, and, in particular, by several aptamers, including the thrombinbinding aptamer tba that. Structural dynamics of thrombinbinding dna aptamer dggttggtgtggttgg quadruplex dna studied by largescale explicit solvent simulations. A special class of thrombin synthetic ligands is represented by aptamers, which are dna or rna oligonucleotides that bind specific target molecules with high affinity and selectivity. The structure of a complex between human alphathrombin and a ggttggtgtggttgg 15nucleotide consensus sequence has been solved by xray crystallography and refined at 2. Matic kovacic, peter podbevsek, hisae tateishikarimata, shuntaro takahashi, naoki sugimoto, janez plavec, thrombin binding aptamer g quadruplex stabilized by pyrenemodified nucleotides, nucleic acids research, volume 48, issue 7.
Selective recognition of parallel and antiparallel. We successfully observed the structural change between the g. Novel modular dna aptamer for human thrombin with high. Despite extensive investigations on gquadruplexes, there is still a need to further. Trapping the gquadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded. Representation of thrombin functional sites, illustration of the creation of the 1. Thrombinrna aptamer, phosphorodithioate rna, ps2rna, ps2 linkage, hydrolaserna complex. One of the major difficulties that arises when selecting aptamers containing a gquadruplex is the correct amplification of the ssdna sequence. Aptamers for diagnostics with applications for infectious. Mapping the affinity landscape of thrombinbinding aptamers on 2. Gquadruplex based dna aptamers for human thrombin are promising pharmaceuticals as anticoagulants.
Anticoagulant, dna aptamer, hemostasis, inhibitor, modular structure, thrombin. A family of dna aptamers with varied duplex region length. The tba sequence comprises 15 deoxynucleotide residues figure 1a and adopts an intramolecular, antiparallel g4 structure with chairlike conformation. Thrombin aptamer selfassembles into a highly stable gquadruplex structure, and thrombin is a structurally stable globular protein found in blood. Figure 1 sequence of the thrombin aptamer and its schematic structure.
Molecular dynamics study of thrombin capture by aptamers tba26 and tba29 coupled to a dna origami. Thrombin binding aptamer, more than a simple aptamer. Dnas form various structures through hydrogenbonding, basestacking and electrostatic interactions. We have used twodimensional 1h nmr spectroscopy to study the conformation of the thrombin binding aptamer d. We have performed thermodynamic analysis, binding affinity and biological activity studies of tba variants modified by unlocked nucleic acid una monomers. Different duplexquadruplex junctions determine the. Dna aptamer configuration affects the sensitivity and.
Another structure that is formed by the oxytricha telomere sequence has a parallel and antiparallel neighbor that results in three different groove widths. Gquadruplexes g4 have been found increasing potential in applications, such as molecular therapeutics, diagnostics and sensing. The bestknown example is the thrombinbinding aptamer, tba, a singlestranded 15mer dna that inhibits the activity of thrombin, the key enzyme of coagulation cascade. Study of the interaction between the gquadruplexforming. A novel gquadruplex motif in the human met promoter region. For example, an electrochemical thrombin aptasensor was constructed by immobilizing a thrombin aptamer tba labeled with redoxactive methylene blue mb on an electrode.
Gquadruplex has the same thermal stability within 15tgt and 31tgt duplex is stable enough. Gquadruplex aptamer targeting protein a and its capability to detect staphylococcus aureus demonstrated by elona. We systematically examined the structural feature of the tba gquadruplex aptamer with fluorine19 19f labels at all of the thymidine positions. Protein c, and the structurally related serine proteases thrombin and activated factor vii.
In this paper, we report studies concerning thrombin binding aptamer. Initially discovered 15mer dna aptamer 15tba has a minimal g. A 15mer dna aptamer named tba adopts a g quadruplex structure that strongly inhibits fibrinclot formation by binding to thrombin. Each immobilized state was detected by observing the change of charge transfer resistance rct in a nyquist plot fig. Simulation of aptamer thrombin complex formation process with funnel metadynamics funnel metadynamics, method from. Lightcontrolled thrombin catalysis and clot formation using a. Una residues may improve gquadruplex anticoagulant properties. Although these noncovalent interactions are known to be cooperative in stabilizing a gquadruplex g4 structure of dna, we find from allatom molecular dynamics simulations that the. The structure of the 31meric aptameric dna oligonucleotide ra36 was studied. Anti thrombin aptamers are g quadruplex bearing oligonucleotides, which recognizes the exosites of human thrombin. Aptamerinduced thermofluorimetric protein stabilization.
The guest is the thrombinbinding aptamer tba that folds into the gquadruplex in the presence of cations. They can be generated using various experimental, theoretical, and computational techniques. Aptamers based on nucleic acids are a promising alternative to antibodies in therapy and diagnostics. Lower k d value of modified aptamers for thrombin binding might not be preferred in certain cases. The dye specifically binds to gquadruplex gq forming nucleic acids, such as the 29mer thrombin binding aptamer hd22 or tba29 and hemin binding aptamer ps2.
Thrombin was known to induce tba to form the antiparallel quadruplex and bind to this structure with high affinity. Folding of the thrombin aptamer into a gquadruplex with. This is the first book to detail bioanalytical technologies and methods that have been developed using aptamers in analytical, medical, environmental, and food science applications. Tba complex has been determined using nmr spectroscopy and restrained molecular dynamics simulations. The oligonucleotide forms a unimolecular dna quadruplex consisting of two gquartets connected by two tt loops and one tgt loop. Beside involvement in the regulation of telomerase activity, gquadruplexes have been linked to proteins that either specifically bind to or promote the folding of quadruplex structures 1, 129. Thermodynamic, anticoagulant, and antiproliferative properties of. In this study, we demonstrated that 19f nmr can be used to study the thrombinbinding aptamer tba dna gquadruplex, widely used as a model structure for studying gquadruplex aptamers. Prothrombin coagulation factor ii is proteolytically cleaved to form thrombin in the clotting process. Ability of thrombin to act as molecular chaperone, inducing formation of quadruplex structure of thrombin binding aptamer. Schematics of detection method and preparation steps for the aptamerbased serrs sensor. Unfolding mechanism of thrombinbinding aptamer revealed. Detailed molecular modelling experiments provided novel insights into the. Another example is the thrombinbinding aptamer that folds into an intramolecular quadruplex with three lateral loops, often depicted as a chair structure.
Tba folds as a gquadruplex structure, as proved by its nmr structure. Furthermore, the xray crystal structure of this aptamer in complex with thrombin was solved showing the gquadruplex aptamer sandwiched between two positively charged regions of two separate. Thrombinbinding dna aptamer forms a unimolecular quadruplex structure in solution james a. In the current work, the interaction between the thrombinbinding aptamer tba, 5. The binding constants of tba mutants correlate with the gquadruplex stabilities with thrombin. As in solution, in the complex the singlestranded dna folds into a structure with two gquartets. In addition, binding competition experiments were performed to find out the binding site of the aptamers on apc. Thrombinbinding aptamer tba is a good example of an oligonucleotide that exploits quadruplex motif for thrombinbinding 1.
Molecular dynamics study of thrombin capture by aptamers. Una on the structural dynamics and stability of tba. After binding thrombin, the tba adopts a gquadruplex structure, which moves mb away from the electrode. Ra36 aptamer has a twopattern structure, which includes two grich regions capable of forming a gquadruplex.
Structural dynamics of thrombinbinding dna aptamer d. The addition of thrombin results in the assembly of the two aptamer subunits into the thrombin aptamer g quadruplex structure, in which the 3. To record and measure the binding of thrombin to gquadruplex chips. The application of a highperformance liquid chromatography. Stability, heat, and hydration it has been shown that the dna aptamer dg2t2g2tgtg2t2g2 adopts an. The structure of alphathrombin inhibited by a 15mer.
Investigating the properties of tba variants with twin thrombin. Antithrombin aptamers are gquadruplexbearing oligonucleotides, which recognizes the. Molecules free fulltext gquadruplexforming aptamers. Future studies to optimize other aptasensors with less stable aptamers and analytes in complex matrices are likely to be challenging. Modular construction of dna aptamers for human thrombin. In a bare capillary, the peak area of gquadruplex aptamer gapt was found to decrease with the addn. The bestknown example is the thrombin binding aptamer, tba, a singlestranded 15mer dna that inhibits the activity of thrombin, the key enzyme of coagulation cascade. The systematic evolution of ligands by exponential enrichment which uses iterative screening of nucleic acid libraries is a popular experimental technique. Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. Structure of gtetrad showing hydrogen bonds between fo.
Novel modular dna aptamer for human thrombin with high anticoagulant activity. Tba1 to its target thrombin and in identifying novel 2. Dna aptamermediated regulation of the hairpin ribozyme by human. Anticoagulant, coagulation, hemostasis, gquadruplex, molecular dynamic simulation, pharmacodynamics, pharmacokinetics, thrombin, thrombin binding dna aptamer, thrombin inhibitor, turbidimetry, antiinflammatory properties. T bp is formed between the two tt loops across the diagonal of the top gquartet. In this study, we investigated the impact of different chemical modifications such as a three. Moduleactivity relationship of gquadruplex based dna. The first anti thrombin aptamer, tba, was generated through via selex systematic evolution of ligands by exponential enrichment technology in 1992 by l. Improved thrombin binding aptamer by incorporation of a. Molecular aptamer beacons for realtime protein recognition jianwei j. The first and the mostwell known anticoagulant aptamer was the thrombin binding aptamer tba, also termed hd1, arc183, discovered by bock in 1992 using selex. Detection for folding of the thrombin binding aptamer.
Can aptamers containing a gquadruplex be selected from a degenerate library using nonequilibrium capillary electrophoresis ce of equilibrium mixtures neceem along with highthroughput illumina sequencing. Thrombin binding aptamer tba is able to inhibit the activity of thrombin. Thrombin binding aptamer gquadruplex stabilized by pyrene. Thrombin stabilizes the gquadruplex via the interaction with residues in the loops. Based on sequence analysis and spectrophotometric studies in solution, a mixed duplexquadruplex structure was proposed for re31 aptamer figure 1c 25,28,32. Dna aptamermediated regulation of the hairpin ribozyme by. In particular, the effect of single incorporation of a novel 4thiouracil derivative.
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