Kinetics of DNA Threading Intercalation by a Rigid Ruthenium Complex Dimer

Abstract

Compounds that can intercalate DNA by threading between melted bases serve as a model of therapeutic drugs for malignant tumors. An essential property of effective antitumor activity requires a drug candidate to exhibit a slow dissociation rate from the intercalation state of DNA-drug complex. In particular, previous bulk experiments reported a very slow dissociation rate for the rigid Ruthenium complex dimer ΔΔ-P (Δ,Δ-[µ-(11,11'-bidppz)(phen)4Ru2]4+). We examined the kinetics of ΔΔ-P threading intercalation into an individual λ-DNA molecule held between a micropipette and an optical trap over a concentration range of 2-100 nM. DNA extensions due to threading intercalation are measured at several concentrations for constant stretching forces of 10-60 pN. As a result, fractional binding is determined as a function of force and concentration from the time-dependent approach to equilibrium extension, which yields equilibrium binding affinity. We also obtain the force and concentration-dependent on and off rates for the threading reaction. Furthermore, these measurements allow us to obtain the force-dependent and zero force binding affinity. By fitting these rates to the expected exponential dependence on force, we are able to extract the equilibrium change in DNA extension due to a single intercalation event as well as the distance to the transition state from the bound and unbound equilibrium states. These results show that the DNA length must increase significantly both for association and dissociation of the ligand, which explains the extremely slow binding kinetics.