Quantifying the molecular mechanism for highly stereo-selective DNA threading intercalation

DNA threading intercalators, such as binuclear ruthenium complexes, are regarded as potential DNA-targeted therapeutic drugs because of slow kinetics and high affinity. Recent bulk studies reported that poly(dAdT) threading intercalation by the binuclear ruthenium complex [$\mu $-dppzip(phen)$_{4}$Ru$_{2}$]$^{4+}$(Piz) is highly stereo-selective. The largest fractional binding was achieved for $\Delta $,$\Lambda $-Piz, with the $\Delta $ (right handed) configuration at the intercalating dipyridophenazine (dppz) subunit and the $\Lambda $ (left handed) configuration at the distal imidazophenanthroline (ip) subunit. To quantify this highly stereo-selective molecular mechanism, we used optical tweezers to probe single $\lambda $-DNA molecules elongation due to the threading intercalation by each of $\Delta $,$\Delta $-Piz and $\Delta $,$\Lambda $-Piz. While maintaining a DNA stretching force of 30 pN and a ligand concentration of 5 nM, the elongation was traced until reaching equilibrium. Then it was traced back to the free DNA extension by rinsing out the bound ligands. We found that the equilibrium elongation for $\Delta $,$\Lambda $-Piz is 30{\%} larger, and the affinity is 50{\%} higher relative to $\Delta $,$\Delta $-Piz. Further force-dependent study will quantitatively determine the differences in the zero-force binding site size, affinity and the DNA structural dynamics for association and dissociation.