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). In this study, we compared properties of RE31 aptamer and its analogues containing varying number of bases in the duplex region and nucleotide insertions in the hinge region. Reduction in the number of nucleotides in the duplex region by 1 to 4 pairs (in comparison with RE31 aptamer) resulted in the decrease of the structural stability of aptamers (manifested as lower melting temperatures) and their ability to inhibit thrombin-stimulated fibrin formation in human blood plasma in tests of thrombin, prothrombin, and activated partial thromboplastin times. However, an increase in the number of bases by 1 to 2 pairs did not cause significant changes in the stability and antithrombin activity of the aptamers. Insertions into the hinge region of RE31 aptamer decreased its antithrombin activity. Investigation of RE31 antithrombotic properties demonstrated that RE31 (i) slowed down thrombin formation in human blood plasma (thrombin generation test), (ii) accelerated lysis of fibrin clot by tissue plasminogen activator in in vitro model, and (iii) suppressed arterial thrombosis in in vivo model. Based on the obtained data, RE31 aptamer can be considered as a potentially effective antithrombotic compound.

[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.

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. In our experiments, the negatively charged aptamer DNA RE31 was coated step-by-step with positively charged protamine. They had protamine/aptamer ratios of 0.2/1 and 0.4/1 by charge, with particle size being determined by dynamic light scattering. The aptamer DNA-protamine complexes were administered to rats, followed by ex vivo analysis of blood samples. The results showed that prothrombin time (PT) increased by a factor of 5.6-6.7 within 2 h after injection and remained at approximately the same level for 6 h, while injections of pure protamine did not lead to any noticeable change in clotting time. Thus, complexation with protamine proved to prolong the inhibitory activity of the RE31 DNA aptamer.

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. Furthermore, the affinity of an aptamer to thrombin and prothrombin increases (respectively from 2.7×10⁻8 M to 5.6×10⁻¹° M and from 1.8×10⁻5 M to 7.1×10⁻9 M) with an increase in the number of nucleotide pairs in the duplex region.

[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). A distinctive feature of the Ec-Lon is its ability to interact with DNA, however either DNA binding site(s) or the role ofthe complex Ec-Lon · DNA have not yet been characterized. A promising tool for the study of molecular mechanisms of interaction between nucleic acids and protein ligands are known to be aptamers (small nucleic acids with high specificity to organic compounds of different nature). Ec-Lon-protease was found to form complexes with the previously obtained thrombin aptamers whose molecules comprise the duplex domains and G-quadruplex region. The aptamer affinities to the enzyme have been characterized. The synthesis of novel aptamers specific to Ec-Lon protease is planed for studying the mechanism of the enzyme-DNA complexation.

[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 1.7 x 10(-10) M depending on the orientation of the aptamer, an insoluble matrix. The sorbents were stable during multiple use. Conditions for sorbent regeneration were picked up. These chromatographic materials can be used for medical and biotechnological applications.

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. In this paper, we applied the circular dichroism and UV spectroscopy to characterize the temperature unfolding and conformational features of 31-mer thrombin-binding aptamer (31TBA), whose sequence has a potential to form G-quadruplex and duplex domains. Both structural domains were monitored independently in 31TBA and in several control oligonucleotides unable to form either the duplex region or the G-quadruplex region. The major findings are as follows: (1) both duplex and G-quadruplex domains coexist in intramolecular structure of 31TBA, (2) the formation of duplex domain does not change the fold of G-quadruplex, which is very similar to that of 15TBA, and (3) the whole 31TBA structure disrupts if either of two domains is not formed: the absence of duplex structure in 31TBA abolishes G-quadruplex, and vice versa, the lack of G-quadruplex folding results in disallowing the duplex domain.

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. On the other hand, RE31 did not reduce amidolytic activity of thrombin towards the short peptide substrate, in other words, did not modify the state of enzyme active center. By the capacity to inhibit clotting reactions, RE31 was superior to the previously described highly effective 31-component antithrombin aptamer 31TBA (thrombin binding aptamer, TBA). The effect of RE31 was species-specific: it inhibited human thrombin activity more effectively than activities of rat and rabbit thrombins.

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. A de novo RNA binding site for heterologous S7 protein of Thermus thermophilus (r6hTthS7) was constructed by selection of a combinatorial RNA library based on E. coli ICR: it has only a single supposed protein recognition site in the region of bifurcation. The SERW technique was used for selection of two intercistronic RNA libraries in which five nucleotides of a double-stranded region, adjacent to the bifurcation, had the randomized sequence. One library contained an authentic AG (-82/-20) pair, while in the other this pair was replaced by AU. A serwamer capable of specific binding to r6hTthS7 was selected; it appeared to be the RNA68 mutant with eight nucleotide mutations. The serwamer binds to r6hTthS7 with the same affinity as homologous authentic ICR of str mRNA binds to r6hEcoS7; apparent dissociation constants are 89 +/- 43 and 50 +/- 24 nM, respectively.

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. High affinity and specific binding of aptamers to particular protein targets make them attractive as direct protein inhibitors. They can inhibit pathogenic proteins and data presented here demonstrate that the idea that nucleic acid aptamers can regulate (inhibit) activity of protein targets has been transformed from the stage of basic developments into the stage of realization of practical tasks.

[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. Strong and specific binding for a given protein target of aptamers make them an attractive class of direct protein inhibitors. They can inhibit pathogenic proteins and it is becoming clear that aptamers have the potential to be a new and effective class of therapeutic molecules.

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. Aptamers also inhibited thrombin-induced human platelet aggregation. The inhibitory effects of 31TBA manifested at lower concentrations than those of 15TBA in all tests. These data indicate that the studied antithrombin DNA aptamers effectively suppress its two key reactions, fibrin formation and stimulation of platelet aggregation, without modifying active center of the thrombin molecule.

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. During shift-down of rRNA synthesis level, free S7 inhibits self-translation by interacting with 96 nucleotides long specific region of streptomycin (str) mRNA between cistrons S12 and S7 (intercistron). Many bacteria do not have the extended intercistron challenging development of specific approaches for searching putative mRNA regulatory regions, which are able to interact with proteins. The paper describes application of SERF approach (Selection of Random RNA Fragments) to reveal regulatory regions of str mRNA. Set of random DNA fragments has been generated from str operon by random hydrolysis and then transcribed into RNA; the fragments being able to bind protein S7 (serfamers) have been selected by iterative rounds. S7 binds to single serfamer, 109 nucleotide long (RNA109), derived from the intercistron. After multiple copying and selection, the intercistronic mutant (RNA109) has been isolated; it has enhanced affinity to S7. RNA109 binds to the protein better than authentic intercistronic str mRNA; apparent dissociation constants are 26 +/- 5 and 60 +/- 8 nM, respectively. Location of S7 binding site on the mRNA, as well as putative mode of regulation of coupled translation of S12 and S7 cistrons have been hypothesized.

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. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.

[Aptameric DNA--a new type of thrombin inhibitor]. / Aptamernye DNK--ingibitory trombina novogo tipa.

The formation of complexes between various thrombin preparations and a 30-mer aptamer DNA was comparatively studied, and a correlation between the complex formation and the fibrinogen-hydrolyzing activity of thrombin was found. The aptamer DNA was shown to inhibit the fibrin formation from fibrinogen.

[Study of the binding of the S7 protein with 16S rRNA fragment 926-986/1219-1393 as a key step in the assembly of the small subunit of prokaryotic ribosomes]. / Izuchenie sviazyvanie belka S7 s fragmentom 926-986/1219-1393 16S rRNK--kliuchevogo étapa sborki maloi subchastitsy prokarioticheskikh ribosom.

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coli and Thermus thermophilus S7 with a fragment of the 3' domain of the E. coli 16S rRNA. Both proteins showed a high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA-protein interactions.

A study of the thermophilic ribosomal protein S7 binding to the truncated S12-S7 intercistronic region provides more insight into the mechanism of regulation of the str operon of E. coli(1).

A study of the ability of His6-tagged ribosomal protein S7 of Thermus thermophilus to interact with the truncated S12-S7 intercistronic region of str mRNA of Escherichia coli has been described. A minimal S7 binding mRNA fragment is a part of the composite hairpin, with the termination codon of the S12 cistron on one side and the initiation codon of the next S7 cistron on the other. It has a length in the range of 63-103 nucleotides. The 63 nucleotide mRNA fragment, which corresponds to a putative S7 binding site, binds very poorly with S7. Tight RNA structure models, which behave as integral systems and link the S7 binding site with the translational regulation region of the hairpin, are suggested. This observation provides more insight into the mechanism of S7-directed autogenous control of translational coupling of str mRNA.