Victor Zozulya

Effect of Ni2+ and Cd2+ ions on thermally induced conformational transitions in poly(dA)-poly(dT) system

Effects of Ni2+ and Cd2+ ions on thermally induced conformational transitions in the poly(dA)·poly(dT) polynucleotide duplex and poly(dA)·2poly(dT) triplex under near physiological ionic conditions were studied by measurement of UV absorption melting curves and static light scattering intensity. The diagrams of conformational transitions in poly(dA)-poly(dT)-Me2+ systems were plotted. An aggregation in these polynucleotide systems arises at certain values of the metal ions concentration and the temperature after the polymer dissociation into single strands. The phenomenon is conditioned by the aggregation of poly(dA) via the interstrand cross-linking by the dication bridges. Unlike Ni2+, Cd2+ induces formation of very stable aggregates which did not disintegrate even upon cooling up to room temperature.

Calculating Helix-to-Coil Transitions of Duplexes Formed by Phenazine-Conjugated Oligonucleotide, Using Flourescence Melting Data

A novel intercalating phenazine derivative (Pzn) was covalently linked to the 3′-end of decathymidylate via a ribose residue of the dye. A fluorescence technique was used to study double helix formation by this conjugate with poly(rA) in aqueous solutions of neutral pH, at the presence of 0.1 and 1 M sodium ions. Proportionality between thermally induced changes in the fluorescence intensity of the free conjugate and bound one was revealed, that made it possible to calculate the helix-to-coil transition from fluorescence melting data using a simple equation. The transition curves were found to be in well conformity with those constructed from absorption measurements. It was shown that the attachment of Pzn significantly enhanced the stability of poly(rA) · (dT)10 duplex due to intercalation of the dye chromophore into the adenine strand. The temperature of half-dissociation was increased by 12°C, and the stabilizing increment of standard free energy was 3.2–3.6 kcal/mol at 37°C.

Interaction of a tricationic meso-substituted porphyrin with guanine-containing polyribonucleotides of various structures

The interaction of a tricationic water-soluble meso-(N-methylpyridinium)-substituted porphyrin, TMPyP3+, derived from classic TMPyP4, with double-stranded poly(G)  ⋅  poly(C) and four-stranded poly(G) polyribonucleotides has been studied in aqueous buffered solutions, pH 6.9, of low and near-physiological ionic strengths in a wide range of molar phosphate-to-dye ratios (P/D). To clarify the binding modes of TMPyP3+ to biopolymers various spectroscopic techniques, including absorption and polarized fluorescence spectroscopy, Raman spectroscopy, and resonance light scattering, were used. As a result, two competitive binding modes were revealed. In solution of low ionic strength outside binding of the porphyrin to the polynucleotide backbone with self-stacking prevailed at low P/D ratios (P/D  <  3.5). It manifested itself by the substantial quenching of porphyrin fluorescence. Also the formation of large-scale porphyrin aggregates was observed near the stoichiometric binding ratio. The spectral changes observed at P/D  >  30 including emission enhancement were supposed to be caused by the embedding of partially stacked porphyrin J-dimers into the polymer groove. TMPyP3+ binding to poly(G) induced a fluorescence increase 2.5 times as large as that observed for poly(G)  ⋅  poly(C). In solution of near-physiological ionic strength the efficiency of external porphyrin binding was reduced substantially due to the competitive binding of Na+ ions with the polymer backbone. The spectroscopic characteristics of porphyrin bound to polynucleotides at different conditions were compared with those for free porphyrin.

Fluorescent Studies on Cooperative Binding of Cationic Pheophorbide-a Derivative to Polyphosphate

The cooperative binding of a novel water‐soluble cationic derivative of pheophorbide‐a (CatPheo‐a) to inorganic polyphosphate (PPS) in buffered aqueous solutions was studied by means of polarized fluorescence spectroscopy in a wide range of molar phosphate‐to‐dye ratios (P/D). Under low P/D values, CatPheo‐a forms extended stacking associates on the PPS matrix, while under high P/D the dye binds to PPS in the dimer form. The CatPheo‐a self‐association is accompanied by 40‐fold dye fluorescence quenching and a substantial increase in the fluorescence polarization degree. The fluorescent titration data were used for determination of cooperative binding parameters by Schwarzs method.

Fluorescence Properties of Intercalating Neutral Chromophores in Complexes with Polynucleotides of Various Base Compositions and Secondary Structures

Changes in the relative quantum yield and polarization degree of steady-state fluorescence of daunomycin (DM) on its binding to six synthetic single- and double-stranded polynucleotides of various nucleotide compositions were measured over a wide range of molar polymer-to-dye ratios, in solutions of low ionic strength. Guanine base was found to be an effective quencher of DM fluorescence [in the DM–poly(G) complex the intensity of residual emission was ~0.5% of the free dye intensity]. The quenching of DM fluorescence by another purine base, i.e., adenine, was also revealed. But, unlike guanine, adenine exhibits quenching activity when it is in close contact with the DM chromophore, as realized in the complex with single-stranded poly(A). In intercalative complexes with double-stranded nucleic acids, where such contact is lacking, the quenching ability of adenine does not manifest itself, which has been demonstrated with the DM–poly(A) · poly(U) complex. It was found that the interaction with pyrimidine bases does not substantially change the DM quantum yield. The quenching feature of DM fluorescence is identical to that observed earlier by us for a glycoside phenazine dye containing, like DM, a neutral chromophore.

Spectroscopic Studies on Binding of Porphyrin-Phenazine Conjugate to Four-Stranded Poly(G)

Binding of a novel cationic porphyrin–imidazophenazine conjugate, TMPyP3+–ImPzn, to four-stranded poly(G) was investigated in aqueous solutions of neutral pH under near physiological ionic conditions using absorption, polarized fluorescent spectroscopy and fluorescence titration techniques. In absence of the polymer the conjugate folds into stable internal heterodimer with stacking between the porphyrin and phenazine chromophores. Binding of TMPyP3+–ImPzn to poly(G) is realized by two competing ways. At low polymer-to-dye ratio (P/D < 6) outside electrostatic binding of the cationic porphyrin moieties of the conjugate to anionic polynucleotide backbone with their self-stacking is predominant. It is accompanied by heterodimer dissociation and distancing of phenazine moieties from the polymer. This binding mode is characterized by strong quenching of the conjugate fluorescence. Increase of P/D results in the disintegration of the porphyrin stacks and redistribution of the bound conjugate molecules along the polymer chain. At P/D > 10 another binding mode becomes dominant, embedding of TMPyP3+–ImPzn heterodimers into poly(G) groove as a whole is occurred.