Abstract
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 technique confirms the ability of two eight ring hairpin-polyamides, with similar architectures but differing at a single ring position (Py to Im), to retain in vitro specificities and display distinct genome-wide binding profiles.
Highlights
Regulating genomic architecture and activity with sequence-specific synthetic DNA binding molecules is a long-standing goal at the interface of chemistry, biology and medicine
Hairpin Py-Im polyamide is conjugated at the C-terminus with a psoralen and biotin for enrichment connected via a linker (~36 Å extended) capable of sampling pyrimidine proximal to the polyamide-binding site suitable for 2 + 2 photocycloaddition [23, 24]
Scatter plot comparison analysis of all enriched 8-mer sequences indicates a preference for the consensus motif, polyamide 1 prefers WGWWCW over WGGWCW, whereas polyamide 2 prefers WGGWCW over WGWWCW, Fig 2C and S3 Fig. These results demonstrate that a single position (CH to N:) modification of the aromatic amino acid ring of the polyamide core structure imparts a significant change in the global in vitro DNA sequence preferences and confirms that the C-terminus modification does not have significant impact on the specificity of the hairpin polyamides
Summary
Regulating genomic architecture and activity with sequence-specific synthetic DNA binding molecules is a long-standing goal at the interface of chemistry, biology and medicine. Small molecules that selectively target desired genomic loci could be harnessed to regulate critical gene networks. The greatest success in designing small molecules with programmable DNAbinding specificity has been with pyrrole-imidazole (Py-Im) polyamides [1,2,3,4,5,6,7,8]. Pyrrole-imidazole (Py-Im) polyamides are synthetic DNA-binding oligomers with high sequence specificity and affinity [7].
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