The Sequence Context and Methylation Dependence of DNA Drug Binding

Abstract

The ability of molecules to bind DNA plays an important role in many biological processes including, transcription, regulation, replication, and repair. Understanding the factors that influence binding affinity is therefore essential to our understanding of DNA. In this study the effects of sequence context and binding-site methylation on the binding affinity and conformation of DNA dodecamers are explored. The Cre binding site (ACGT) was studied in its methylated and native forms with a number of different flanking sequences chosen to test the predictive power of a previously published dinucleotide scale, wherein certain dinucleotides were said to have an affinity for either the BI or BII DNA conformation. 31P-NMR was used to test the true conformational state of each phosphorous in the dodecamers, and it was found that the scale could not accurately predict the real conformation of DNA. Importantly, the conformation of the binding site, the central tetrad which was not changed, varied significantly based on the sequence context. Thus more complex relationships than dinucleotides, such as longer-range translational conformation effects, are shown to affect the DNA conformation. Fluorescence titration with the DNA intercalator 7-Aminoactinomycin D was performed to determine the KD of each of the dodecamers and provide biological context to our conformation results. Excellent binding affinities were seen in the ACAC, ACAG, and GGCC sequences, while weaker binding was observed for GGAG sequences. The effects of methylation on binding affinity and conformation demonstrated that methylation effects are modulated by sequence context.