G-Tetrad-Selective Ligand Binding Kinetics in G-Quadruplex DNA Probed with Fluorescence Correlation Spectroscopy.

Abstract

Probing the kinetics of ligand binding to biomolecules is of paramount interest in biology and pharmacology. Measurements of such kinetic processes provide information on the rate-determining steps that control the binding affinity of ligands to biomolecules, thereby predicting the mechanism of the molecular interaction. In this context, ligand binding to G-quadruplex DNA (GqDNA) structures has attracted tremendous attention primarily because of their use in possible anticancer therapy. Although a large number of G-quadruplex-specific ligands have been proposed, probing the kinetics of G-tetrad-selective binding of (multiple) ligands within a G-quadruplex DNA (GqDNA) structure remains challenging. Most of the earlier studies focused on the thermodynamics of ligand binding; however, the kinetics of ligand association and dissociation with GqDNA, particularly binding of multiple ligands within a GqDNA structure, have not been explored. Here, we propose a simple fluorescence correlation spectroscopy-based method that measures the G-tetrad-selective association and dissociation rates of ligands within a GqDNA structure by correlating the fluorescence fluctuations of a site-specific (5' or 3' end-labeled) fluorophore (Cy3) in GqDNA due to quenching of Cy3 fluorescence, induced by the ligand binding to the G-tetrads. We show that well-known GqDNA ligands, BRACO19, TMPyP4, Hoechst 33258, and Hoechst 33342, have G-tetrad-selective association and dissociation rates, which suggest site-dependent variation of free energy barriers for binding/unbinding of the ligands with GqDNA. We also show that the measured kinetic rates depend not only on the G-tetrad site (5' vs 3' end) but also on the ligand and GqDNA structures.