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

Background: The ability to target specific DNA sequences using small molecu les has major implications for basic research and medicine. Previous studie s revealed that a bis-intercalating molecule containing two 1,4,5,8-napthal enetetracarboxylic 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-intercalat ing molecules that have partially randomized peptide linkers. Results: A library of bis-intercalating derivatives with varied peptide lin kers was screened for sequence specificity using DNase I footprinting on a 231 base pair (bp) restriction fragment. The library mixtures produced foot prints that were generally similar to the parent bis-intercalator, which bo und within a 15 bp G + C-rich repeat above 125 nM. Nevertheless, subtle dif ferences in cleavage enhancement bands followed by library deconvolution re vealed a derivative with novel specificity. A lysine-tris-beta-alanine deri vative was found to bind preferentially within a 19 bp palindrome, without substantial loss of affinity. Conclusions: Synthetically simple changes in the bis-intercalating compound s can produce derivatives with novel sequence specificity. The large size a nd symmetrical nature of the preferred binding sites suggest that cooperati vity may be retained despite modified sequence specificity. Such findings, combined with structural data, could be used to develop versatile DNA ligan ds of modest molecular weight that target relatively long DNA sequences in a selective manner.