Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single molecule techniques, we quantify the binding of a small metalorganic ruthenium threading intercalator (Δ,Δ-B), and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinity is the same for both molecules, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, these kinetics are still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that binding affinity is modulated independently by the intercalating section and binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a “lock mechanism”, in which a large length increase of the DNA duplex is required both for association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association, but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association, but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared to conventional intercalation and will pave the way for rational design of therapeutic drugs based on DNA threading intercalation