Preferential Binding of Epirubicin Hydrochloride with Single Nucleotide Mismatched DNA and Subsequent Sequestration by a Mixed Micelle

Targeting mismatched base pairs containing DNA using small molecules and exploring the underlying mechanism involved during the binding interactions is one of the fundamental aspects of drug design. These molecules in turn are used in nucleic acid targeted therapeutics and cancer diagnosis. In this work, we systematically delineate the binding of the anticancer drug, epirubicin hydrochloride (EPR) with 20-mer duplex DNA, having both natural nucleobase pairing and thermodynamically least stable non-Watson-Crick base pairing. From the thermal denaturation studies, we observed that EPR can remarkably enhance the thermal stability of cytosine-cytosine (CC) and cytosine-thymine (CT) mismatched (MM) DNA over other 20-mer duplex DNA. From steady-state fluorescence spectroscopy and isothermal titration calorimetry studies, we concluded that EPR binds strongly with the mismatched duplex DNA through the intercalation binding mode. The interaction of EPR and duplex DNA has also been monitored at a single molecular resolution using fluorescence correlation spectroscopy (FCS). Dynamic quantitates such as diffusion coefficients and hydrodynamic radii obtained from an FCS study along with association and dissociation rate constants estimated from intensity time trace analyses further substantiate the stronger binding affinity of EPR to the thermally less stable mismatched DNA, formed by the most discriminating nucleobase (viz. cytosine). Additionally, we have shown that EPR can be sequestered from nucleic acids using a mixed micellar system of an anionic surfactant and a triblock copolymer. From thermal denaturation studies and circular dichroism spectroscopy, we found that the extent of drug sequestration depends on the binding affinity of EPR to the duplex DNA, and this mixed micellar system can be employed for the removal of excess drug in the case of a drug overdose.