Single position substitution of hairpin pyrrole-imidazole polyamides imparts distinct DNA-binding profiles across the human genome

Regulating desired loci in the genome with sequence-specific DNA-binding molecules is a major goal for the development of precision medicine. Pyrrole-imidazole (Py-Im) polyamides are synthetic molecules that can be rationally designed to target specific DNA sequences to both disrupt and recruit transcriptional machinery. While in vitro binding has been extensively studied, in vivo effects are often difficult to predict using current models of DNA binding. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their effective deployment in vivo. In this report we identified polyamide-DNA interaction sites across the entire genome, by covalently crosslinking and capturing these events in the nuclei of human LNCaP cells. This method, termed COSMIC-seq, confirms the ability of hairpin-polyamides, with similar architectures but differing at a single ring position, to retain in vitro specificities and display distinct genome-wide binding profiles. These results underpin the development of Py-Im polyamides as DNA-targeting molecules that mediate their regulatory or remedial functions at desired genomic loci.