Correlating Drug Binding Affinities with Base Pair Opening Rates in DNA

Abstract

The cyclic AMP responsive element (Cre) is a highly conserved stretch of DNA that is involved in the activation of gene transcription. Our group has studied the drug binding of a fluorescent derivative of a DNA intercalating anti-cancer drug, 7-amino actinomycin D (7-AMD), to the Cre sequence in different sequence contexts. We initially analyzed the DNA backbone conformation of Cre samples with varying flanking sequences and correlated these values to the binding affinities of 7-AMD. These studies revealed several anomalies that suggest that the conformation described by BI/BII content of the DNA backbone is, at most, only partially responsible for the 7-AMD binding affinity to the Cre sequence. This result is not surprising as 7-AMD has a conjugated ring structure in addition to its peptidyl side chains which interact with the backbone. As a result, we began studying DNA base pair opening rates and correlating these with 7-AMD binding affinities to account for the intercalation of 7-AMD into the Cre sequence. DNA base pair opening rates were determined by tracking imino proton exchange via NMR spectroscopy in the presence of a varying concentration of base catalyst. In this study, both two-dimensional NOESY and one-dimensional H1 NMR spectroscopy are used to track the change in line widths of the imino protons of the central Cre binding site for five sequences with varying flanking sequences. Trends in opening rates reveal that sequences with strong 7-AMD binding feature slower base pair opening dynamics than sequences with weaker 7-AMD binding. These results suggest that local base stacking is important for 7-AMD binding, a hypothesis we are currently investigating with UV spectroscopy.