Erwin Graham S, Grieshop Matthew P, Bhimsaria Devesh, Eguchi Asuka, Rodríguez-Martínez José A, Ansari Aseem Z
Department of Biochemistry, University of Wisconsin-Madison.
Department of Biochemistry, University of Wisconsin-Madison; Department of Electrical and Computer Engineering, University of Wisconsin-Madison.
J Vis Exp. 2016 Jan 20(107):e53510. doi: 10.3791/53510.
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.
基因组是一些最有效的化疗药物的作用靶点，但这些药物大多缺乏DNA序列特异性，这会导致剂量限制性毒性和许多不良副作用。用序列特异性小分子靶向基因组可能会产生治疗指数更高、脱靶效应更少的分子。N-甲基吡咯/N-甲基咪唑聚酰胺是一类能够经过合理设计以极高精度靶向特定DNA序列的分子。与大多数天然转录因子不同，聚酰胺能够结合甲基化和染色质化的DNA，且亲和力不会降低。聚酰胺的序列特异性已在体外通过同源位点鉴定（CSI）以及传统的生化和生物物理方法进行了广泛研究，但关于聚酰胺在细胞中与基因组靶点结合的研究仍然难以捉摸。在此，我们报告一种方法——小分子交联分离染色质（COSMIC），该方法可识别全基因组范围内的聚酰胺结合位点。COSMIC与染色质免疫沉淀（ChIP）类似，但在两个重要方面有所不同：（1）使用光交联剂以实现对聚酰胺结合事件的选择性、时间可控捕获，（2）利用生物素亲和手柄在半变性条件下纯化聚酰胺-DNA缀合物，以减少非共价结合的DNA。COSMIC是一种通用策略，可用于揭示聚酰胺和其他靶向基因组的化疗药物在全基因组范围内的结合事件。
