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

• Published in: Journal of molecular structure 2020-08, Vol.1214, p.128218, Article 128218
• Main Authors: Ryazanova, Olga A., Zozulya, Victor N., Voloshin, Igor M., Dubey, Larysa V., Ilchenko, Mykola M., Dubey, Igor Ya, Karachevtsev, Victor A.
• Format: Article
• Language: English
• Subjects: Binding; Fluorescent light-up probe; G-quadruplex; Pheophorbide–phenazinium conjugate; Polyribonucleotides
• ISSN: 0022-2860; 1872-8014
• DOI: 10.1016/j.molstruc.2020.128218

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. [Display omitted] •New conjugate of Pheophorbide-a with cationic phenazinium dye has been synthesized.•Its emission is quenched in aqueous medium as intramolecular heterodimer is formed.•DFT data show that heterodimer in water is energetically more stable than open form.•Conjugate binding to poly(G) quadruplex results in up to 50-fold emission increase.•Pheo-Pzn allows to discriminate between duplex and quadruplex polynucleotides.