Binding of Pheophorbide-a methyl ester to nucleic acids of different secondary structures: A spectroscopic study

Abstract

Binding of neutral Pheophorbide-a methyl ester (MePheo-a) to various synthetic polynucleotides, double-stranded poly(A)-poly(U), poly(G)-poly(C) and four-stranded poly(G), as well as to calf thymus DNA, was studied using the methods of absorption and polarized fluorescent spectroscopy. Measurements were performed in aqueous bufferes solutions (pH 6.9) of low ionic strength (2 mM Na+) in a wide range of molar phosphate-to-dye ratios (P/D). Absorption and fluorescence characteristics of complexes formed between the dye and biopolymers were determined. Binding of MePheo-a to four-stranded poly(G) is shown to be accompanied by the most significant spectral transformations: hypochromism of dye absorption,large batochromic shift of Soret absorption band (~26 nm) and fluorescence band (~9 nm) maxima, 48-fold enhancement of the dye emission intensity. In contrast, its binding to the double-stranded polynucleotides and native DNA induces only small shifts of absorption and fluorescence bands, as well as no more than 4-fold rise of fluorescence intensity. Substantial spectral changes and high value of fluorescence polarization degree (0.26) observed upon binding of MePheo-a to quadruplex poly(G) allow us to suggest the intercalation of the dye chromophore between guanine tetrads. At the same time, small spectral changed and insignificant increase of MePheo-a fluorescence polarization degree (0.12) upon binding of the dye to double-stranded biopolymers point to another binding type. Incorporation of MePheo-a to a helix groove is supposed to occur, presumably in the dimeric form. Substantial enhancement of MePheo-a emission upon binding to four-stranded poly(G) allows us to propose this compouns as a new fluorescent probe G-quadruplex structures.