Peptide bis-intercalator binds DNA via threading mode with sequence specific contacts in the major groove.

BACKGROUND: We previously described a general class of DNA polyintercalators in which 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) intercalating units are connected via peptide linkers, resulting in the first known tetrakis- and octakis-intercalators. We showed further that changes in the composition of the peptide tether result in novel DNA binding site specificities. We now examine in detail the DNA binding mode and sequence specific recognition of Compound 1, an NDI bis-intercalator containing the peptide linker gly-gly-gly-lys. RESULTS: 1H-NMR structural studies of Compound 1 bound to d(CGGTACCG)(2) confirmed a threading mode of intercalation, with four base pairs between the diimide units. The NMR data, combined with DNAse I footprinting of several analogs, suggest that specificity depends on a combination of steric and electrostatic contacts by the peptide linker in the floor of the major groove. CONCLUSIONS: In view of the modular nature and facile synthesis of our NDI-based polyintercalators, such structural knowledge can be used to improve or alter the specificity of the compounds and design longer polyintercalators that recognize correspondingly longer DNA sequences with alternating access to both DNA grooves.

An octakis-intercalating molecule.

Herein we report the synthesis and characterization of a polyintercalator with eight potential intercalating l,4,5,8-naphthalenetetracarboxylic diimide (NDI) units linked in a head-to-tail arrangement via a peptide linker. UV spectroscopy and viscometry measurements indicated the molecule binds to double-stranded DNA with all eight NDI units intercalated simultaneously. Competition dialysis and DNAse 1 footprinting studies revealed a preference for GC-rich regions of DNA, and circular dichroism studies revealed significant distortion of B-form DNA upon binding. Our so-called "octamer" represents, to the best of our knowledge, the first intercalator that binds as an octakis-intercalator, capable of spanning at least 16 base pairs of DNA.

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.