Thermodynamics and Kinetics of Unfolding of Antiparallel G-Quadruplexes in Anti-Thrombin Aptamers.

The process of unfolding of G-quadruplex structure in the RE31 DNA-aptamer and in its complex with thrombin under the action of the fluorescently labeled complementary oligonucleotides of varying length with formation of double-helix structures has been studied. It has been suggested that G-quadruplex unfolding involves formation of an intermediate complex with an oligonucleotide. Thermodynamic parameters and kinetics of unfolding of the free aptamer and its complex with thrombin differ.

DNA Aptamers to Thrombin Exosite I. Structure-Function Relationships and Antithrombotic Effects.

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin receptors on platelet surface, thereby suppressing thrombin-stimulated formation of fibrin from fibrinogen and platelet aggregation. Earlier, we synthesized original antithrombin aptamer RE31 (5'-GTGACGTAGGTTGGTGTGGTTGGGGCGTCAC-3') that contained (in addition to G-quadruplex) a hinge region connected to six pairs of complementary bases (duplex region).

[Study of the quantity of interleukin-6 by SDS-PAAG electrophoresis and immuno-enzyme analysis in mixed saliva after rinsing the oral cavity with oligonucleotide specific.].

The selective properties of a solution of oligonucleotide specific to IL-6 on the concentration of IL-6 in mixed saliva of patients with oral inflammatory processes were studied using SDS-PAGE by electrophoresis and enzyme immunoassay. The application of these methods showed that in the mixed saliva of patients after rinsing with a solution of an oligonucleotide specific for IL-6, the amount of IL-6 decreases. The ELISA Kit and 20% SDS-PAGE showed the highest sensitivity to determine the concentration of IL-6 in saliva, which should be considered in clinical laboratory practice.

Four steps for revealing and adjusting the 3D structure of aptamers in solution by small-angle X-ray scattering and computer simulation.

Nucleic acid (NA) aptamers bind to their targets with high affinity and selectivity. The three-dimensional (3D) structures of aptamers play a major role in these non-covalent interactions. Here, we use a four-step approach to determine a true 3D structure of aptamers in solution using small-angle X-ray scattering (SAXS) and molecular structure restoration (MSR).

Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer.

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer.

Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers.

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172.

Complex formation with protamine prolongs the thrombin-inhibiting effect of DNA aptamer in vivo.

Antithrombin DNA aptamersRE31 are single-chain oligonucleotides that fold into three-dimensional forms allowing them to bind the enzyme with high affinity and inhibit its activity in vivo. They are rapidly degraded by a nonspecific nuclease, and, to prolong the lifetime of the aptamer DNA in the bloodstream, it is necessary to coat it with a polymer envelope. A new approach to solving this problem based on preparation of DNA-polyelectrolyte complexes with a minimal particle size that can circulate with blood flow.

[Interaction of DNA Aptamers with the ATP-Dependent Lon Protease from Escherichia coli].

ATP-dependent Lon protease of E. coli (Ec-Lon) is a key enzyme of the quality control system of the cell proteome. Ec-Lon subunit comprises N-terminal non-catalytic region, ATPase module and proteolytic domain (serine-lysine endopeptidase).

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.

A family of DNA aptamers with varied duplex region length that forms complexes with thrombin and prothrombin.

Structural properties determine binding affinities of DNA aptamers specific to thrombin. Our paper is the first to focus on a family of eight G-quadruplex-based aptamers with varied duplex region length (from two to eight base pairs). We have shown that the duplex, which is not the main binding domain, greatly influences the interaction with thrombin and prothrombin.

[DNA aptamer based sorbents for binding human IgE].

DNA aptamer based sorbents are synthesized for binding human IgE. Sorbents effectively removed IgE from human blood plasma. The experimental values of IgE desorption constants were from 11 x 10(-l0) to 1.

Thrombin inhibitors based on single-stranded DNA aptamers.

Investigation of inhibitory effect of two single-stranded DNA thrombin-inhibiting aptamers (15TBA and 31TBA) on fibrin polymerization in fibrinogen solutions and comparison of anticoagulant properties of these aptamers by a new global coagulation test of thrombodynamics. Measurement of aptamers' functional stability in human plasma and blood in vitro in order to investigate the involvement of 3'-exonuclease in fast decrease of aptamers' functional activity in vivo. Thrombin inhibition activity was measured in a buffer system in vitro as effects of aptamers on fibrin polymerization.

Coexistence of G-quadruplex and duplex domains within the secondary structure of 31-mer DNA thrombin-binding aptamer.

A number of thrombin-binding DNA aptamers have been developed during recent years. So far the structure of just a single one, 15-mer thrombin-binding aptamer (15TBA), has been solved as G-quadruplex. Structures of others, showing variable anticoagulation activities, are still not known yet.

Characteristics of a new DNA aptamer, direct inhibitor of thrombin.

Characteristics of a new antithrombin DNA-aptamer RE31 were studied. This aptamer inhibited thrombin formation in human plasma catalyzed by exogenous (lengthening of thrombin time) and endogenous thrombin (lengthening of partial prothrombin time and activated partial thromboplastin time). In addition, the aptamer completely suppressed thrombin-induced aggregation of human platelets.

[Molecular recognition elements--DNA/RNA-aptamers to proteins].

In this review summarizes data on DNA/RNA aptamers--a novel class of molecular recognition elements. Special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. These include aptamers to serine protease, to cytokines/growth factors, to influenza viral protein, nucleic acid binding proteins.

Structural Dynamics of Thrombin-Binding DNA Aptamer d(GGTTGGTGTGGTTGG) Quadruplex DNA Studied by Large-Scale Explicit Solvent Simulations.

The thrombin-binding aptamer (15-TBA) is a 15-mer DNA oligonucleotide with sequence d(GGTTGGTGTGGTTGG). 15-TBA folds into a quadruplex DNA (G-DNA) structure with two planar G-quartets connected by three single-stranded loops. The arrangement of the 15-TBA-thrombin complex is unclear, particularly with respect to the precise 15-TBA residues that interact with the thrombin structure.

Mapping the ribosomal protein S7 regulatory binding site on mRNA of the E. coli streptomycin operon.

In this work it is shown by deletion analysis that an intercistronic region (ICR) approximately 80 nucleotides in length is necessary for interaction with recombinant E. coli S7 protein (r6hEcoS7). A model is proposed for the interaction of S7 with two ICR sites-region of hairpin bifurcations and Shine-Dalgarno sequence of cistron S7.

Molecular recognition elements: DNA/RNA-aptamers to proteins.

The review summarizes data on DNA/RNA aptamers, a novel class of molecular recognition elements. Special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. These include aptamers to serine proteases, cytokines, influenza viral proteins, immune deficiency virus protein and nucleic acid binding proteins.

[Physiological and hygienic characteristic of high-precision manufacturing operations in microelectronics].

It was shown that workers performing high-precision manufacturing operations in microelectronic industry undergo severe visual, nervous and emotional stress combined with significant locomotor load, air deionization and deozonation, bacterial contamination, and UV deficit at their workplaces. These working conditions promote development of negative changes in the visual analyzer, nervous and emotional disorders, disturbances of systemic and regional hemodynamics. Also impaired is the functional state of the upper limb neuromuscular apparatus, central nervous and cardiovascular systems.

Inhibition of thrombin activity with DNA-aptamers.

The effects of two DNA aptamers (oligonucleotides) 15TBA and 31TBA (15- and 31-mer thrombin-binding aptamers, respectively) on thrombin activity were studied. Both aptamers added to human plasma dose-dependently increased thrombin time (fibrin formation upon exposure to exogenous thrombin), prothrombin time (clotting activation by the extrinsic pathway), and activated partial thromboplastin time (clotting activation by the intrinsic pathway). At the same time, these aptamers did not modify amidolytic activity of thrombin evaluated by cleavage of synthetic chromogenic substrate.

Selection of random RNA fragments as method for searching for a site of regulation of translation of E. coli streptomycin mRNA by ribosomal protein S7.

In E. coli cells ribosomal small subunit biogenesis is regulated by RNA-protein interactions involving protein S7. S7 initiates the subunit assembly interacting with 16S rRNA.

Preparation of functionally active recombinant human interleukin-6.

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture.

Mapping contacts of the S12-S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli.

In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the 'streptomycin' operon transcript by binding to the S12-S7 intercistronic region.