Study of complexation of single-stranded poly(rA) and poly(rU) with methylene blue.

To reveal the effect of DNA- or RNA-specific low-molecular compounds on cellular processes on the molecular level, we have carried out the studies with the application of spectroscopic methods. It is necessary for the understanding of structural-functional properties of nucleic acids in cell. In this work the interaction of DNA-specific thiazine dye methylene blue (MB) with synthetic polynucleotides poly(rA) and poly(rU) was studied. The interaction of MB with synthetic polyribonucleotides poly(rA) and poly(rU) was examined in the solution with high ionic strength in a wide phosphate-to-dye (P/D) range, using the absorption and fluorescence spectroscopies, as well as the fluorescence 2D spectra and 3D spectra analyses were given. Values of the fluorescence quenching constants for the complexes of MB with poly(rA) and poly(rU) were calculated (KSV is the Stern-Volmer quenching constant). Two different modes of MB binding to single-stranded (ss-) poly(rA) and poly(rU) and to their hybrid double-stranded (ds-) structure - poly(rA)-poly(rU) were identified. This ligand binds to ss-poly(rA) and poly(rA)-poly(rU) by semi-intercalation and electrostatic modes, but to ss-poly(rU) the prevailing mode is the electrostatic interaction.Communicated by Ramaswamy H. Sarma.

Study of complexes of Hoechst 33258 with poly(rA)-poly(rU) depending on various ionic strengths in the water-saline solution.

Comparative study of the complexes of groove-binding ligand Hoechst 33258 (H33258) with synthetic homopolynucleotides poly(rA)-poly(rU) and poly(dA)-poly(dT) has been carried out at various concentration ratios of r = ligand/nucleic acids (NA) and different ionic strengths of the water-saline solution 0.02, 0.04 and 0.1 M, using the method of UV-melting. It was revealed that the melting curves of the complexes of poly(dA)-poly(dT) with H33258 at the low concentrations of ligand are biphasic, which actually does not depend on the solution ionic strength. In the case of the complexes of poly(rA)-poly(rU)-H33258, the melting curves become quasi-biphasic only at the ionic strength 0.02 M and relatively high concentrations of the ligand. Differential melting curves (DMC) of the mentioned polynucleotides and their complexes with H33258 were obtained as well. DMC of poly(rA)-poly(rU) were found to be significantly wide at the ionic strengths of the solution 0.02 and 0.04 M and to show an intrinsic heterogeneity of double-stranded structure of this polynucleotide.Communicated by Ramaswamy H. Sarma.

Study of interaction of methylene blue with DNA and albumin.

The interaction of thiazine dye methylene blue (MB) with Calf thymus DNA and human blood serum albumin (HSA) has been studied. MB was revealed to stabilize the native structure of DNA and HSA, since the melting temperature of the complexes is shifted to higher values in relation to that of both macromolecules in pure state. It was also revealed that the absorption and fluorescence spectra of the MB-DNA complexes change significantly, while those of MB-albumin complexes do not change noticeably. Analysis of the obtained data allows to conclude that MB binds to DNA by two modes, including intercalation and electrostatic mechanisms. In the case of HSA, the main binding mode of MB, conditioning the stabilization of the protein native structure, is the electrostatic mechanism.Communicated by Ramaswamy H. Sarma.

Hybridization kinetics of DNA fragments in the presence of ligands intercalating into DNA-duplexes.

In this work the hybridization kinetics of DNA fragments on underlayer has been studied, when besides single-stranded DNA molecules there are also ligands in the medium that are able to intercalate into DNA-duplexes. A system of differential equations was obtained that describes the correlative change of the number of DNA-duplexes on the underlayer and the change of the number of ligands intercalating into DNA-duplexes. It was shown that the rate of underlayer filling by DNA-duplexes increases along with the enhancement of both equilibrium constant of formation reaction of DNA-duplexes and the concentration of DNA targets in the solution. It was also shown that the intercalation kinetics of ligands into DNA-duplexes relevantly depends on relation of dissociation rate constant of DNA-duplex to dissociation rate of the ligand complex with DNA-duplex.Communicated by Ramaswamy H. Sarma.

Spectroscopic study of interaction of various GC-content DNA with Hoechst 33258 depending on Na+ concentration.

Communicated by Ramaswamy H. Sarma.

Transient decondensation of chromatin in liver nuclei of rats treated with tannic acid.

Communicated by Ramaswamy H. Sarma.

Study of the influence of the ionic strength on complex-formation of ethidium bromide with poly(rA)-poly(rU).

Communicated by Ramaswamy H. Sarma.

Theoretical treatment of helix-coil transition of complexes DNA with two different ligands having different binding parameters.

The melting transition of DNA-ligand complexes, allowing for two binding mechanisms to different DNA conformations is treated theoretically. The obtained results express the behavior of the experimentally measurable quantities, degree of denaturation, and concentrations of bound ligands on the temperature. The range of binding parameters is obtained, where denaturation curves become multiphasic. The possible application to the nanocomposites crystallization is discussed.

Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters.

Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.

Kinetics of adsorption of extended ligands on DNA at small fillings.

In the present work, the adsorption kinetics of extended ligands on DNA duplexes at small fillings when molecules of DNA duplexes are on the underlayer within diffusion layer has been investigated. Both diffusion of ligands in solution (diffusion stage) and adsorption of ligands (kinetic stage) are taken into consideration at adsorption of ligands on DNA duplexes. Nonlinear system of differential equations describing adsorption of ligands where not only diffusion stage but also kinetic stage is taken into account, is obtained, moreover the equations allow localizing duplexes in arbitrary place within diffusion layer. Numeric solution of the equations makes possible to investigate the filling kinetics of DNA duplexes by ligands depending on parameters controlling adsorption process. It has been shown that depending on relation between adsorption parameters different kinetic regimes of adsorption - kinetic, complex, and diffusion regimes may be realized.

Influence of ionic strength on Hoechst 33258 binding with DNA.

The binding of Hoechst 33258 with DNA at various ionic strengths of solution and different ligand concentrations has been investigated. Existence of more than one type of interactions of Hoechst 33258 with DNA has been revealed, which were very sensitive to the ionic strength. Hoechst 33258 doesn't show specificity to AT sequences of DNA at low ionic strength. High affinity binding mode becomes obvious at high ionic strength. The values of binding constants and binding site sizes for revealed strong and weak interactions have been determined.

Complex-formation of Ethidium Bromide with poly[d(A-T)].poly[d(A-T)].

The interaction of Ethidium Bromide (EtBr) with double-stranded (ds-) and single-stranded (ss-) poly[d(A-T)] was studied in different ionic strengths solutions. Optical spectroscopy and Scatchard analysis results indicate that the ligand interacts to both helix and coiled structures of the polynucleotide by "strong" and "weak" binding modes. The association parameters (binding constant -K- and the number of nucleotides corresponding to a binding site -n) of the strong type of interaction were found to be independent of Na+ concentration. Weak interaction occurs at low ionic strength and/or high EtBr concentration. Estimated binding parameters of EtBr with ss- and ds-polynucleotide are in good agreement with those for EtBr-B-DNA complexes. Data obtained provided an evidence for a stacking interaction of EtBr with single stranded poly[d(A-T)].