Mapping Polyamide–DNA Interactions in Human Cells Reveals a New Design Strategy for Effective Targeting of Genomic Sites

Abstract

Targeting the genome with sequence-specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high-affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.

Synthetic genome readers target clustered binding sites across diverse chromatin states

“…We named this approach cross-linking of small molecules to isolate chromatin (COSMIC) (31). COSMIC employs trifunctional derivatives of polyamides that…”

Deciphering the Genomic Targets of Alkylating Polyamide Conjugates Using High-Throughput Sequencing

“…The pellet of nuclei was carefully resuspended in modified binding buffer (22) Control experiments were performed without PIP-indoleseco-CBI conjugates and with a 0.1% final concentration of DMSO. We used the PIP concentrations from the previous report (22) that were consistent with the PIP quantity measured in the nuclei of treated cells (34). PIP-containing nuclei were washed with micrococcal nuclease (MNase) buffer (10 mM Tris-HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine and 0.1× protease inhibitor cocktail) (30).…”

“…Briefly, samples were washed 5 min once with 0.5 ml of washing buffer 1 (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 3% SDS), once with altered washing buffer 2 (10 mM TrisCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% NP40), 2× with altered washing buffer 3 (10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 0.1% NP-40) and 3× with TE. The samples were then resuspended in elution buffer (10 mM Tris-HCl (pH 7.6), 0.4 mM EDTA and 100 mM KOH) (22) and DNA was eluted from magnetic beads after heating at 90 • C for 30 min. The remaining DNA with the beads were eluted using elution buffer 2 (2% SDS, 100 mM NaHCO 3 and 3 mM biotin) with heating to 65 • C for 8-12 h. The detached samples were purified with a QIAquick PCR purification Kit (Qiagen, CA, USA) and quantified.…”

“…The complex organization of chromatin packaging in the nucleus may be a critical factor for the PIP-binding preferences along the genome (19)(20)(21). A recent report by the Ansari group (22) has initiated the preliminary effort to map the PIPbinding sites in the human genome by developing an approach called 'crosslinking of small molecules for isolation of chromatin' or COSMIC, thereby directing the evolution in PIP design strategy for effectively targeted gene regulation. Data provided by ChIP-seq regarding the genomewide mapping of key transcription factors and regulatory element-binding sites are helpful in the derivation of transcriptional regulation models that govern normal and diseased cell states (23,24).…”

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

“…[25][26][27][28][29] Sequence specificities of Py-Im polyamides have also been investigated by using high-throughput sequencing. [30][31][32] Here, we designed and synthesized thirteen Py-Im polyamides ( Fig.…”