Two-Step DNA Intercalation by Threading of the Flexible Ruthenium Dimer Studied by the Single Molecule DNA Stretching

Abstract

Ruthenium complexes are small synthetic molecules with a wide range of possible uses, including cancer therapy and sensitive fluorescent markers for duplex DNA binding. 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)2dppz2+ moieties connected by a flexible linker. However, the mechanism by which these molecules interact with DNA is not well understood. To quantify these interactions, 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)2dppz 2+ moieties intercalate between base pairs via a threading. We find that the ligand association can only be described by a two-exponential process, indicating multi-step binding. By measuring the concentration dependence of the fast and slow binding modes at several forces and fitting this dependence to a three state kinetic model, we show that the fast mode is bimolecular intercalation of the first dppz moiety, in pre-equilibrium to the ∼10-fold slower intercalation of the second dppz moiety of the same flex-Ru2 molecule. We characterize force-dependence of each rate and DNA elongations associated with each transition state. We estimate the zero-force binding kinetics and equilibrium binding constants for each of the two intercalations steps and of the complete binding process by extrapolating our measured force dependence of these parameters to the force-free state. We conclude that at zero force the [μ-C4(cpdppz)2(phen)4Ru2]4+ binding involves fast (∼20 s) association, slow (∼600 s) dissociation, and very tight (Kd∼10 nM) binding. The methodology developed in this work will be useful for studying other slowly intercalating ligands and proteins.