Abstract
DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[μ‐bidppz‐(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes.
Highlights
When synthesizing DNA-targeted drugs, high DNA binding affinity and slow DNA binding kinetics are considered essential aspects that enhance their therapeutic capability [1,2,3,4,5]
Such nonthreading processes may include pre-intercalation binding to dsDNA, ligand rearrangements due to DNA-DNA contacts, as well as post-intercalation DNA-ligand interactions that do not contribute to DNA elongation
DNA threading by, -P is dramatically facilitated by force, as indicated by the exponential decrease in the DNA-ligand equilibrium dissociation constant with applied stretching force (Figure 3B)
Summary
When synthesizing DNA-targeted drugs, high DNA binding affinity and slow DNA binding kinetics are considered essential aspects that enhance their therapeutic capability [1,2,3,4,5]. Higher affinities maximize the treatment effect with minimal dose exposure, lower association rates enable selective binding molecules to scan DNA for a specific targeted sequence, and lower dissociation rates ensure disruption of DNA transcription and duplication [1,3,4,6,7,8,9] These desired properties are found in ligands that intercalate DNA by threading [10,11,12,13], an interaction that first requires nonintercalating moieties of a ligand to pass between DNA base pairs before the intercalative binding occurs [6,7,14,15]. The equilibrium dissociation constant for the DNA- , -P complex and the molecular mechanism governing the threading intercalation have not been quantitatively determined, due to the limitations
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