Structure and Dynamics of Distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at Low Drug: DNA Ratios

Abstract

Two-dimensional NMR has been used to study the interaction of distamycin A with d(CGCAAA- TTGGC):d(GCCAATTTGCG) at low and intermediate drug: DNA ratios (<2.0). Drug-DNA contacts were identified by nuclear Overhauser effect spectroscopy, which also served to monitor exchange of the drug between different binding sites. At low drug: DNA ratios (0.5), distamycin A binds in two orientations within the five central A-T base pairs and has a preference (2.2:1) for binding with the formyl end directed toward the 5′ side of the A-rich strand. The pattern of drug-DNA contacts corresponding to the preferred binding orientation are consistent with the drug sliding between adjacent AAAT and AATT binding sites at a rate that is fast on the NMR time scale. Similarly, the pattern of NOEs associated with the less favored orientation are consistent with the drug sliding between adjacent AATT and ATTT sites, again in fast exchange. Off-rates for the drug from the major and minor binding orientations were measured to be 2.4 =1.5 and 3.3 = 1.5 s^?1 respectively, at 35°C. At intermediate drug: DNA ratios (1.3) exchange of the drug between the two one-drug and the two sites of a two-drug complex is observed. Off-rates for both drugs from the 2:1 complex were measured to be 1.0 =0.5 s^?1 (35°C).