Structural Transitions in Complementary G-Rich and C-Rich Strands and Their Mixture at Various pH Conditions.

We used circular dichroism spectroscopy, UV spectrophotometry, and differential scanning calorimetry to investigate pH-dependent structural transitions in an equimolar mixture of complementary G-rich d[5'-A(GGGTTA)3GGG-3'] (TelG) and C-rich d[3'-T(CCCAAT)3CCC-5'] (TelC) human telomeric DNA strands. Our studies were conducted at neutral (pH 7.0) and slightly acidic (pH 5.5 and 6.5) pH. We analyzed the melting thermodynamics of TelG and TelC and their equimolar mixture. Our analysis revealed that the preferred conformation of an equimolar mixture of TelG and TelC is the duplex. At pH 5.5, however, in addition to the duplex state, we observed a significant population of the i-motif state formed by TelC. Our results are consistent with the picture in which an increase in pH from 5.5 to 7.0 has little effect on the melting enthalpy of an isolated G-quadruplex while causing a strong reduction in the melting enthalpy of an isolated i-motif (the latter diminishes to 0 at pH 7.0). These effects summarily lead to a decrease in the contribution of the i-motif to the melting enthalpy of the mixture and, hence, an increase in the apparent melting enthalpy and overall stability of the duplex state.

Interaction of MnTOEtPyP4 porphyrin with DNA.

The binding of water-soluble meso-tetra-(4N-oxyethylpyridyl) porphyrin (H2TOEtPyP4) and its manganese (III) derivative (MnTOEtPyP4) with calf thymus DNA have been quantitatively studied using UV/Vis spectrophotometry, Circular Dichroism (CD), thermal melting curves and viscometry. The results show, that porphyrins interact with DNA via one binding mode at low relative concentrations (r) and two binding modes at high values of r. The binding constant (Kb) and stoichiometry (n) were determined from binding isotherms for both porphyrin-DNA complexes. The thermal melting analysis indicates that the double-helical structure of DNA molecules is stabilizing in presence of studied porphyrins. At certain concentrations of porphyrin, two-stage melting curves were observed, which indicates the existence of two different binding modes. Obtained results show that MnTOEtPyP4 associates with DNA duplex via outside binding mode.Communicated by Ramaswamy H. Sarma.

The structural features of poly(dG-dC).poly(dG-dC) at complexation with some porphyrins.

The binding peculiarities of the water-soluble meso-tetra-(4N-hydroxyethylpyridyl) porphyrin (H2TOEtPyP4) and its Cu- and Co-derivatives (CuTOEtPyP4 and CoTOEtPyP4) with synthetic double-stranded alternating polynucleotide poly(dG-dC).poly(dG-dC) were investigated by UV/Vis absorption and circular dichroism (CD) methods. It was shown that the porphyrins with planar structure such as H2TOEtPyP4 and CuTOEtPyP4 interact with poly(dG-dC).poly(dG-dC) via intercalation at low relative concentrations (r = [porphyrin]/[polynucleotide]), while at high r - via intercalation and external binding modes. In the case of no planar porphyrin CoTOEtPyP4 complexation occurs only by external binding mode. The binding constant Kb and the exclusion parameter n calculated for H2TOEtPyP4, CuTOEtPyP4 and CoTOEtPyP4 porphyrins with poly(dG-dC).poly(dG-dC) complexes was 1.50 x107, M-1 (n = 1.76); 9.29 x107, M-1 (n = 1.18); and 0.28 x107, M-1 (n = 2.65) correspondingly. The values of binding parameters for each porphyrin-poly(dG-dC).poly(dG-dC) complexes demonstrated good agreement with the proposed binding models. Communicated by Ramaswamy H. Sarma.

pH-dependent complex formation of Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin with cancer DNA.

The complex formation between the synthetic water-soluble Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin (ZnTOEPyP4) and cancer DNA in comparison to healthy DNA was investigated using the UV/VIS spectrophotometry method in phosphate-buffered saline at different pHs. The increasing of DNA/porphyrin ratio leads to hypochromicity and red shift in the Soret band, which indicate the complexation of the ZnTOEPyP4 with DNA. The results show that the binding constant (Kb) and the exclusion parameter (n) of ZnTOEPyP4 with DNA strongly depend upon the pH. The Kbof ZnTOEPyP4 with cancer DNA is higher than with normal DNA.Communicated by Ramaswamy H. Sarma.

Effect of Urea on G-Quadruplex Stability.

G-quadruplexes represent a class of noncanonical nucleic acid structures implicated in transcriptional regulation, cellular function, and disease. An understanding of the forces involved in stabilization and destabilization of the G-quadruplex conformation relative to the duplex or single-stranded conformation is a key to elucidating the biological role of G-quadruplex-based genomic switches and the quest for therapeutic means for controlled induction or suppression of a G-quadruplex at selected genomic loci. Solute-solvent interactions provide a ubiquitous and, in many cases, the determining thermodynamic force in maintaining and modulating the stability of nucleic acids. These interactions involve water as well as water-soluble cosolvents that may be present in the solution or in the crowded environment in the cell. We present here the first quantitative investigation of the effect of urea, a destabilizing cosolvent, on the conformational preferences of a G-quadruplex formed by the telomeric d[A(G3T2A)3G3] sequence (Tel22). At 20 mM NaCl and room temperature, Tel22 undergoes a two-state urea-induced unfolding transition. An increase in salt mitigates the deleterious effect of urea on Tel22. The urea m-value of Tel22 normalized per change in solvent-accessible surface area, ΔSA, is similar to those for other DNA and RNA structures while being several-fold larger than that of proteins. Our results suggest that urea can be employed as an analytical tool in thermodynamic characterizations of G-quadruplexes in a manner similar to the use of urea in protein studies. We emphasize the need for further studies involving a larger selection of G-quadruplexes varying in sequence, topology (parallel, antiparallel, hybrid), and molecularity (monomolecular, bimolecular, tetramolecular) to outline the advantages and the limits of the use of urea in G-quadruplex studies. A deeper understanding of the effect of solvent and cosolvents on the differential stability of the G-quadruplex and duplex conformations is a step toward elucidation of the modulating influence of different types of cosolvents on duplex-G-quadruplex molecular switches triggering genomic events.

Two-photon microscopy imaging of oxidative stress in human living erythrocytes.

Red blood cells (RBCs) are known to be the most suitable cells to study oxidative stress, which is implicated in the etiopathology of many human diseases. The goal of the current study was to develop a new effective approach for assessing oxidative stress in human living RBCs using two-photon microscopy. To mimic oxidative stress in human living RBCs, an in vitro model was generated followed by two-photon microscopy imaging. The results revealed that oxidative stress is clearly visible on the two-photon microscopy images of RBCs under oxidative stress compared to no fluorescence in controls (P<0.0001). This novel approach for oxidative stress investigation in human living RBCs could efficiently be applied in clinical research and antioxidant compounds testing.

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes.

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.