Influence of DNA base sequence on the binding energetics of actinomycin D

Abstract

The influences of base sequence on the thermodynamic properties associated with the interaction of actinomycin D with DNA are examined. It has been previously established that GpC steps of double-helical DNAs are highly preferred binding sites for actinomycin D. In this study, a series of oligonucleotides was designed and synthesized to probe the effects of flanking base sequence (adjacent to the GpC step) and novel non-GpC binding sites on the binding of actinomycin D. The use of these oligonucleotides provides a direct method for quantitating sequence specificities and actinomycin D binding energetics. Effects of different 5' and 3' flanking nucleotides on the interactions of actinomycin with the core GpC binding sites were examined using UV-visible spectrophotometric methods, and changes in binding energetics were quantitated. These studies demonstrate strong actinomycin D binding affinities to both classical GpC and an atypical non-GpC site. Enthalpy and entropy components of the DNA binding energetics for the GpC binding sites are compared and correlated with those determined for actinomycin D binding to the high-affinity non-GpC site of an 11-mer containing TGGGT as the central sequence. This TGGGT site, first suggested to be a high-affinity sequence in our earlier photoaffinity labeling studies, exhibits binding of actinomycin D comparable in strength to that of traditional actinomycin D binding sites (i.e., GpC steps). From these studies, the overall affinity and specific thermodynamic contributions (delta H degree, delta S degree) to binding of actinomycin D are demonstrated to be highly influenced both by the sequence at the intercalation site and by neighboring bases which flank the intercalation site.