Quantifying the DNA Binding Kinetics of a Ruthenium Dimer Complex with a Flexible Linker

Abstract

Ruthenium complexes are small synthetic molecules with a wide range of possible uses, including cancer therapy, and as a research tool to understand chemical carcinogenesis. The ruthenium complex [μ-C4(cpdppz)2(phen)4Ru2]4+ has been engineered to have a high affinity for DNA and a low dissociation rate. The complex consists of two Ru(phen)2+ moieties connected by a flexible linker. Strong binding inhibits replication of DNA in target cancer cells. To quantify the rate at which DNA-ligand binding occurs, double-stranded DNA is stretched with optical tweezers, and exposed to the ligand under a fixed applied force. When binding to DNA, the two Ru(phen)2+ moieties intercalate between base pairs via a threading mechanism. Intercalation results in an increase in the length of the target DNA, and this change depends exponentially upon the applied force. Separate fast and slow binding modes are revealed, indicating multi-step binding. Both the fast and the slow rates are dependent on force, and each binding event contributes equally to the extension change. The concentration dependence reveals that the fast mode is bimolecular, while the slow mode is unimolecular. We are also able to quantify the on and off rates of each mode at a given force with a three state kinetic model. These studies demonstrate the capability of optical tweezers to elucidate the mechanism of complex DNA-ligand interactions, which may facilitate the rational design of DNA binding ligands with specific DNA interaction properties.