Pheophorbide–phenazinium conjugate as a fluorescent light-up probe for G-quadruplex structure

Abstract

A novel fluorescent light-up probe for G-quadruplex (G4) structures based on fluorescence quenching mechanism was designed. It is a conjugate of two fluorophores, Pheophorbide-a (Pheo-a) and cationic intercalative aminophenazinium dye (Pzn), linked with polymethylene chain. Pheo-a is a natural porphyrin known as efficient photosensitizer for antitumor photodynamic therapy. Cationic Pzn part of the conjugate enhances its binding to anionic nucleic acid. Spectroscopic properties of Pheo-Pzn and its binding to polyribonucleotides of different secondary structures (double-stranded poly(A)·poly(U), poly(G)·poly(C) and four-stranded poly(G)) were studied in a wide range of molar phosphate-to-dye ratios (P/D) by absorption spectroscopy, polarized fluorescence and fluorimetric titration methods. In aqueous medium free Pheo-Pzn exhibits strongly quenched fluorescence due to formation of stacked intramolecular heterodimer. DFT calculations demonstrated that it is energetically favored over the open form of Pheo-Pzn. The conjugate is able to discriminate between duplex and quadruplex polynucleotides exhibiting 50-fold emission increase upon binding to quadruplex poly(G) and quenched fluorescence in the case of ds-polymers. The observed spectral changes evidence disintegration of the heterodimer and separate binding of both Pheo-Pzn fragments to poly(G) with intercalation of Pheo-a moiety between guanine tetrads, while binding of the heterodimer as a whole to duplex polynucleotides is suggested. Pheo-Pzn conjugate can be proposed as efficient probe for the recognition of G-quadruplex structures.