COSMIC-seq of bioactive hairpin N-methylpyrrole/N-methylimidazole polyamides

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 de-signed 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 binding energetics. Determining the impact of genomic architecture and the local chromatin landscape on polyamide-DNA sequence specificity remains an unresolved question that impedes their utility in vivo. In this report we directly identified polyamide–DNA interaction sites across the entire genome, by covalent 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 and the potential for utility as synthetic transcription factors (Syn-TFs) capable of functioning in concert with the cellular regulatory circuitry. To directly map polyamide binding sites across the genome in LNCaP cells, we utilized COSMIC-seq (crosslinking of small molecules to isolate chromatin). COSMIC consists of treating live cells with trifunctional derivatives of polyamides. These molecules were comprised of a DNA-binding polyamide, an affinity handle, and a photocrosslinker. The polyamide confers sequence specificity, while the affinity handle, biotin, is used to purify polyamide–DNA adducts with streptavidin-coated magnetic beads. The photocrosslinker, psoralen, enables the temporally-controlled formation of a covalent bond between the polyamide and DNA. Cells were first treated with the polyamide derivative, crosslinked with 365 nm UV light, lysed, sonicated to shear DNA, and streptavidin-coated magnetic beads were used to capture polyamide-DNA adducts. Because psoralen crosslinks are reversible, the captured genomic DNA can be separated from the polyamide, purified, and identified by massively parallel, next-generation sequencing (NGS). Sequencing reads are mapped to their location across the genome, and loci bound by polyamides show enrichment of sequencing reads relative to a reference input sample.