Spectroscopic Studies on Binding of Porphyrin-Phenazine Conjugate to Intramolecular G-Quadruplex Formed by 22-mer Oligonucleotide

The interaction of the novel cationic porphyrin – imidazophenazine conjugate (TMP3-Pzn), as well as its zinc (II) and manganese (III) metallocomplexes  with intramolecular G-quadruplex formed by 22-mer oligodeoxynucleotide d[AGGG(TTAGGG)3] of human telomeric repeat (22G4) was studied.  Techniques of absorption and polarized fluorescent spectroscopy as well as absorption melting at 295 nm were used. The binding affinity of the conjugates to the quadruplex was studied using titration experiments registering the dependence of the fluorescence intensity and polarization degree for porphyrin moieties  on quadruplex/ligand molar ratio (P/D). All investigations were carried out in 2 mM phosphate buffer (pH6.9) containing 0.5 mM EDTA and 0.1 M NaCl. Spectroscopic characteristics of the TMP3-Pzn and ZnTMP3-Pzn conjugates evidence the formation of internal heterodimers with stacking between porphyrin and phenazine chromophores, although in the case of MnTMP3-Pzn it has not occurred. It was established that all the conjugates reveal high binding affinities to the intra-molecular G-quadruplex, probably, via external groove binding of porphyrin moiety. The complex formation is accompanied by the increase in the fluorescence intensity and polarization degree of porphyrin fragments. It should be noted that negligibly small fluorescence of manganese porphyrin moiety flames up by near 20 times under the binding, being the novel observation for manganese complex. It was found that ZnTMP3-Pzn and MnTMP3-Pzn stabilize the G-quadruplex structure increasing the transition midpoint temperature by 3 °C, whereas non-metallated TMP3-Pzn conjugate decreases it by approximately 2 °C. Despite of the opposite changes in G-quadruplex melting temperature, a simultaneous increase in the transition enthalpy and entropy was determined for all conjugates, which gives a some stabilizing increment in the Gibbs standard free energy. In the case of metal complexes, 10-fold increase in the 22G4 folding equilibrium constant at 37 °C was determined