Investigating Binding Mechanisms of Small Molecules to Quadruplex DNA

Abstract

Guanine quadruplexes (G4) are dynamic structures found naturally in guanine-rich DNA. When folded, they have been found to block the expression of genes related to diseases including cancer1 and are therefore of significant interest in the field of biomedical research. Our research group has developed a family of chemical binders which strongly stabilize folded G4. This project focuses on quantifying the interaction of these binders with various quadruplexes, through their binding constant (a measure of relative preference for the bound vs unbound state). This constants can be determined through the use of a Fluorescence Intercalation Displacement (FID) biophysical assay,2 a method which measures the competitive binding of the binder molecule and a fluorescent probe to the quadruplex (see figure 1). As a preliminary step, we perform direct titrations of the fluorescent probe, Thiazole Orange (TO), with quadruplex DNA, to determine its binding constant and TO:DNA stoichiometry. TO fluoresces only when bound to a substrate,3 allowing us to track the formation of the TO*DNA complex through an increase in fluorescence. In the FID titration, however, the binder displaces the fluorescent probe, and a decrease in fluorescence intensity is observed. We also discuss mathematical methods used to fit the experimental data in order to determine binding constants for both the fluorescent probe and our binders. A robust model should give evidence in support of the expected binding phenomena.
 References
 1. Hurley, L. et. al. The Febs Journal, 2010, 277, 3459-3469 2. Monchaud, D. et. al. Biochimie, 2008, 90: 1209-1223 3. Yaron, D. et. al. The Journal of Physical Chemistry A. 2008, 112, 9692-9701