Yeva B. Dalyan

Interactions of Meso-tetra-(4-N-oxyethylpyridyl) Porphyrin, Its 3-N Analog and Their Metallocomplexes with Duplex DNA

Abstract Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins.

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex.

Abstract Thermodynamic parameters of melting process (δHm, Tm, δTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage Tm by 18°C, and Tm of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by ∼ 30–40°C. Invariability of ΔHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔTm caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT∼TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.

Melting of complexes of DNA-cis-DDP in acidic environment

The peculiarities of helix-coil transition of DNA at complex-formation with cis-diamminedichlorplatinum (II) (cis-DDP) have been investigated in acidic environment by UV absorbance technique. It is shown that observed features of behavior ΔT (pH) and Tm (pH) DNA at pH 2.8-3.0 are possible to explain by formation in DNA of pseudo-ring structures at covalent linking of cis-DDP with DNA.

The stability of DNA-porphyrin complexes in the presence of Mn(II) ions.

The complex formation of porphyrins with DNA leads to changes of stability of DNA. In the present study we investigated binding properties and the thermodynamic parameters of a water-soluble, cationic planar Cu(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CuTButPyP4] and nonplanar Co(II)-containing meso-tetrakis(4-N-butyl-pyridiniumyl)porphyrin [CoButPyP4] with calf thymus DNA in the presence of divalent manganese ions. For displaying the changes of thermodynamic parameters (T(m) and ΔT) the melting curves of DNA-porphyrin complexes in the presence of Mn(2+) ions have been obtained. The enthalpy (ΔH) of helix-coil transition has been also evaluated. It was shown that the binding of ions to DNA proceeds in two stages depending on the manganese/DNA phosphates molar ratio [Mn]/[P]. At the first stage (0.001<[Mn]/[P]<1), the interaction of manganese ions with DNA phosphates occurs, causing an additional screening of their negative charge and the stabilization of the double helix. As a result, the best conditions for intercalation of CuTButPyP4 or of peripheral rings of CoButPyP4 occur. The significant increase of T(m), but less changes of ΔT were observed. At the second stage (1<[Mn]/[P]<4), the ions interact with both the phosphates and the nitrogen bases of DNA. At this stage, it is possible for the manganese ion to coordinate simultaneously to the oxygen atom of the phosphate and the neighboring base of DNA. At a higher [Mn]/[P] ratio, the destabilization of the double helix begins, and partial breakage of the hydrogen bonds between the nitrogen bases occurs. Respectively the destabilization of DNA in the presence of both porphyrins takes place.

86 Characterization and differentiation of binding modes of water-soluble porphyrins at complexation with DNA

The influence of water-soluble cationic meso-tetra-(4 N-oxyethylpyridyl)porphyrin (H2TOEPyP4) and it’s metallocomplexes with Ni, Cu, Co, and Zn on hydrodynamic and spectral behavior of DNA solutions has been studied by UV/Vis absorption and viscosity measurement. It was shown that the presence of planar porphyrins such as H2TOEPyP4, NiTOEPyP4, and СuTOEPyP4 leads to an increase in viscosity at relatively small concentrations, and then decrease to stable values. Such behavior is explained by intercalation of these porphyrins in DNA structure because the intercalation mode involves the insertion of a planar molecule between DNA base pairs which results in a decrease in the DNA helical twist and lengthening of the DNA. Further decrease of viscosity is explained by the saturation intercalation sites and occurs outside the binding mode. But, in the case of porphyrins with axial ligands such as CoTOEPyP4 and ZnTOEPyP4, the hydrodynamic parameters decrease, which is explained by self-stacking of these porphyrins in DNA surface. This data are proved by spectral measurements. The results obtained from titration experiments were used for calculation of binding parameters: the binding constant K b and the number of binding sites per base pair n. Obtained data reveal that K b varies between 3.4 and 5.4 × 106 M−1 for a planar porphyrins, a range typical for intercalation mode interactions, and 5.6 × 105 M−1 and 1.8 × 106 M−1 for axial porphyrins. In addition, the exclusion parameter n also testifies that at intercalation, (n∼2) the adjacent base pairs are removed to place the planar molecules, and for outside binders to pack on the surface needs too few places (n∼0.5–1). It is apparent that the binding is somewhat stronger at intercalation. The viscometric and spectrophotometric measurements are in good agreement.

Peculiarities of interaction of porphyrins with tRNA at low ionic strength.

Abstract The interaction of meso-tetra-(4N-oxyethylpyridyl)porphyrin (TOEPyP4) and its Zn(II)-, Cu(II)-, Mn(III)-derivatives with tRNA from E.Coli at low ionic strength (μ=0.02M) was studied using UV/Vis spectrophotometry and Circular Dichroism (CD) methods. An unusual Induced Circular Dichroism (ICD) spectra profile of the ZnTOEPyP4-tRNA complex is found. It is demonstrated that ZnTOEPyP4 is ordered in a stack, not only on helical sites, but also on loops of a hairpin form of tRNA. TOEPyP4 and CuTOEPyP4 are able to intercalate in the helical sites of this form of tRNA. MnTOEPyP4 interacts with tRNA via external non-ordered mechanism. It is established that all porphyrins are bound with tRNA more strongly than with DNA.

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.

Thermodynamics of interaction of meso-tetra-(4N-oxyethylpyridyl) porphyrin and its Cu(II)- and Co(II)- containing derivatives with A and B forms of DNA

The interaction of water soluble meso-tetra-(4N-oxyethylpyridyl) porphyrin (TOEPyP4) and its Cu(II)- and Co(II)-containing derivatives (CuTOEPyP4 and CoTOEPyP4) with A and B forms of DNA at low ionic strength was studied via UV-vis spectrophotometry and Circular Dichroism. It is shown that the binding constant of TOEPyP4 and CuTOEPyP4 with A–DNA is two times larger than with B–DNA, and the binding constant of CoTOEPyP4 does not depend on the form of DNA. The thermodynamical analysis based on spectral data indicates the preferable entropic character of porphyrins binding with both forms of DNA. This result shows that at low ionic strength the external groove binding mode is a preferred binding mechanism of these porphyrins with both forms of DNA.

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.

135 The comparative study of H2TOEPyP4 and CoTOEPyP4 porphyrins with A-DNA.

The peculiarities of binding the water-soluble mesotetra-(4N-oxyethylpyridyl) porphyrin (H2TOEPyP4) and its Co-containing form (CoTOEPyP4) with ADNA have been studied by UV–vis spectrophotometry and circular dichroism(CD) methods. All studies were performed in diluted solution of BPSE buffer (BPSE = 6 mM Na2HPO4 + 2 mM NaH2PO4 + 185 mM NaCl + 0.1 mM EDTA), pH 7.0, ionic strength [Na] = 10 M. The results indicated that the presence of Co in porphyrin ring was decreasing binding constant 10 times as compared with H2TOEPyP4 porphyrin. The induced CD spectra was shown (Pasternack, 2003), that H2TOEPyP4 and CoTOEPyP4 porphyrins interact with A-DNA only through external groove binding, while H2TOEPyP4 interact with B-DNA by intercalative mode at small porphyrin–DNA ratio (Barkhudaryan, Ananyan, & Dalyan, 2013), which consequently changes into outside stacking mode at higher rations (Avetisyan, 2014). From the dependencies of binding constants (ln Kb) versus 1/T we evaluated the free binding energies of complexation (ΔGb), enthalpies (ΔHb) and entropies (ΔSb). The increasing entropy (ΔSb) shown that the nature of interaction of porphyrin with A-DNA is predominately hydrophobic. Therefore H2TOEPyP4 and CoTOEPyP4 porphyrins interact with A-DNA through external groove binding.