Altered sequence specificity identified from a library of DNA-binding small molecules

Background: The ability to target specific DNA sequences using small molecules has major implications for basic research and medicine. Previous studies revealed that a bis-intercalating molecule containing two 1,4,5,8-napthalenetetracarboxylic diimides separated by a lysine-tris-glycine linker binds to DNA cooperatively, in pairs, with a preference for G + C-rich sequences. Here we investigate the binding properties of a library of bis-intercalating molecules that have partially randomized peptide linkers. Results: A library of bis-intercalating derivatives with varied peptide linkers was screened for sequence specificity using DNase I footprinting on a 231 base pair (bp) restriction fragment. The library mixtures produced footprints that were generally similar to the parent bis-intercalator, which bound within a 15 bp G + C-rich repeat above 125 nM. Nevertheless, subtle differences in cleavage enhancement bands followed by library deconvolution revealed a derivative with novel specificity. A lysine-tris-beta-alanine derivative was found to bind preferentially within a 19 bp palindrome, without substantial loss of affinity. Conclusions: Synthetically simple changes in the bis-intercalating compounds can produce derivatives with novel sequence specificity. The large size and symmetrical nature of the preferred binding sites suggest that cooperativity may be retained despite modified sequence specificity. Such findings, combined with structural data, could be used to develop versatile DNA ligands of modest molecular weight that target relatively long DNA sequences in a selective manner.