Ensemble and single molecule FRET analysis of the structure and unfolding kinetics of the c-kit promoter quadruplexes

Abstract

FRET analysis has been used to examine the folded conformations and differing kinetic stabilities of two DNA G-quadruplexes (c-kit 1 and c-kit 2) derived from sequences found in the promoter of the c-kit proto-oncogene.

Fig. 1

Schematic for the unfolding of c-kit 1 and c-kit 2 quadruplexes.

Fig. 2

Single molecule histograms of FRET efficiencies for c-kit 1 and c-kit 2 quadruplexes (a) in 0 mM K⁺ for c-kit 1; (b) in 100 mM K⁺ for c-kit 1 with a Gaussian fit; (c) in 0 mM K⁺ for c-kit 2; (d) in 100 mM K⁺ for c-kit 2 with a Gaussian fit. All are in 10 mM Tris HCl (pH 7.4) at 20 °C.

Fig. 3

(a) The decrease of Cy5 fluorescence monitored at 660 nm as the quadruplex formed by kit 1-Cy5:overhang-comp-Cy3 is opened then trapped by kit 1-comp at 50 °C. The data is shown as open circles and fit to a single exponential as a solid line. (b) Arrhenius plot of rate with varying temperature with linear fit of the data for both c-kit 1 (thick dashed line) and c-kit 2 (thin dashed line).