G-Quadruplex Folding Observed by two Photon Fluorescence Correlation Spectroscopy and Dual Time Scale

Abstract

G-rich DNA sequences are known to fold upon addition of salt into a stacked well defined configuration called a quadruplex. A fluorescently labeled 5′-FAM −24-mer G-quadruplex sequence was used to explore the variation of diffusion coefficients at extremely low, low and high KCl concentrations. We found a shift in the diffusion coefficient of about 10μm2/sec toward faster diffusion from extremely low to high KCl concentrations. This shift can be related to the compact structure formed by the G-quadruplex. We have also used a fluorescent guanosine analog, 6MI, to label a 24mer that has shown folding behavior at high KCl concentrations. To explore this further, we have added in excess a sequence that complements the G-rich region to deter the formation of the G-quadruplex. The diffusion coefficient also increased from the unfolded, low KCl concentration to the high salt, G-quadruplex structure. We have constructed a dual-timescale (ps TCSPC and uS-mS FCS) photon correlation system and we are using it to explore linked changes in the fluorophores' lifetimes and the translational diffusion coefficients as they move between low and high salt environments. Part of this work was supported by NIH SCORE Grant S06 GM 060654.