Resolving the DNA Binding Mode of a Rotationally Flexible Binuclear Ruthenium Complex

Abstract

Binuclear ruthenium complexes demonstrate slow dissociation upon binding to DNA, and they are considered as potential DNA-targeted therapeutic drugs. Quantitative investigations of their properties, such as binding affinity, binding mode and kinetics, require approaches beyond traditional experimental techniques. In particular, previous reports showed that the rotationally flexible binuclear ruthenium complex Δ,Δ-[µ-bipb(phen)4Ru2]4+ (Δ,Δ-Pi) strongly condenses DNA. However, the strong DNA condensation poses a challenge in bulk experiments such that even the DNA-ligand binding mode could not be resolved. We examined the kinetics of ΔΔ-Pi interactions with single λ-DNA molecule as a function of a constant applied force of 30 pN and ligand concentration of 5 nM using dual-beams optical tweezers. We find that Δ,Δ-Pi exhibits characteristics of threading intercalation into DNA base pairs. DNA elongation measurements, as monitored over tens of minutes, illustrate two distinct phases during association; rapid intercalation that is analogous to classic intercalation, followed by very slow intercalation that approaches equilibrium with a rate that is comparable to that observed for other threading intercalators. We investigated ΔΔ-Pi dissociation after reaching the equilibrium extension by rinsing the binding ligands from the surrounding solution, and observed that the DNA-ligand complex extension decreases to the DNA-only extension over a timescale longer than the association process. Interestingly, the dissociation measurements fit well to a single rate that is slower than the dissociation rate measured for the previously reported binuclear ruthenium complex Δ,Δ-[µ-bidppz-(phen)4Ru2]4+ (Δ,Δ-P), which is a threading intercalator. Further measurements of force-dependent intercalation thermodynamics and kinetics will allow us to fully quantitatively characterize the complex binding mechanism of this unusual intercalator.