Preferential Site Binding of Monovalent Cations With the Random Coil Form of DNA

Abstract

Fluorescence spectroscopy studies of 7-Aminoactinomycin D (7-AMD) binding to dodecamers containing the Cre binding sequence demonstrate that binding affinity is directly related to both sequence context and cytosine methylation. Importantly, it was also found that methylation effects on binding affinity are sequence dependent. The native d[TTTCACGTGAAA] 2 and d[AA GAACGTTCTT] 2 samples show very similar binding affinities in their native forms, while native d[GAAAACGTTTTC] 2 has a dissociation constant fourfold higher than either of these samples. Upon methylation, d[TTTCA-m 5 -CGTGAAA] 2 shows the greatest change in the dissociation constant, increasing two-fold. The d[AAGAA-m 5 -CGTTCTT] 2 sequence has a 1.5-fold increase in the dissociation constant, while the d[GAAAACGTTTTC] 2 sequence shows no significant change in binding affinity upon methylation. NMR and FTIR evidence will be presented to further explain these phenomena and provide evidence that backbone conformations due to flanking sequence and methylation state are responsible for these differences.