Fluorescence anisotropy and FRET studies of G-quadruplex formation in presence of different cations

Results of the steady-state fluorescence, anisotropy and FRET measurements of G-quadruplex formation in the presence of selected cations (Li+, Na+, K+, NEt4+ and Mg2+) are reported. Three different fluorescent oligonucleotides with human telomeric sequence labeled with fluorescein (FAM) and tetramethylrhodamine (TAMRA) were investigated: a dual-labeled 21-mer denoted as PSO (Potassium Sensing Oligonucleotide) and two 5′- and 3′- single-labeled probes, FAM-21 and 21-TAMRA, respectively. The fluorescence signal of FAM-21 increased significantly for all systems and the fluorescence enhancement was comparable in magnitude for monovalent cations but it was more pronounced for Mg2+ cation. This phenomenon was attributed to the protolytic equilibria of FAM affected by the variation in ionic strength. On the other hand, fluorescence of TAMRA was enhanced selectively by Na(I) cation that was explained by the dequenching of TAMRA emission originated from the peculiarity of the basket-type structure of Na(I)-quadruplex. Anisotropy of FAM-21 (but not 21-TAMRA) appeared to be sensitive to the G-quadruplex formation, showing significant increase with an increase in cation concentration and indicating some restrictions in rotational depolarization of FAM. FRET experiments revealed that all tested cations caused quenching of FAM fluorescence in PSO, but only Na+ and K+ ions produced sensitized emission of TAMRA acceptor. Higher FRET efficiency observed in the presence of sodium ion was attributed to the specific spectral factor and steric interactions in the basket-type Na(I)-quadruplex.