Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

J Vis Exp. 2016 Jan 20:(107):e53510. doi: 10.3791/53510.

Abstract

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Video-Audio Media

MeSH terms

  • Binding Sites
  • Chromatin / chemistry
  • Chromatin / isolation & purification*
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism
  • Genome-Wide Association Study / methods*
  • Humans
  • Imidazoles / chemistry
  • Nylons / chemistry*
  • Nylons / metabolism
  • Pyrroles / chemistry
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / metabolism

Substances

  • Chromatin
  • Imidazoles
  • Nylons
  • Pyrroles
  • Small Molecule Libraries
  • DNA
  • N-methylpyrrole
  • 1-methylimidazole