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Programmable Protein-DNA Crosslinking for the Direct Capture and Quantification of 5-Formylcytosine
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2019-06-04 , DOI: 10.1021/jacs.9b01432
Mario Gieß 1 , Álvaro Muñoz-López 1 , Benjamin Buchmuller 1 , Grzegorz Kubik 1 , Daniel Summerer 1
Affiliation  

5-Formylcytosine (5fC) is an epigenetic nucleobase of mammalian genomes that occurs as intermediate of active DNA demethylation. 5fC uniquely interacts and reacts with key nuclear proteins, indicating functions in genome regulation. Transcription-activator-like effectors (TALEs) are repeat-based DNA binding proteins that can serve as probes for the direct, programmable recognition and analysis of epigenetic nucleobases. However, no TALE repeats for the selective recognition of 5fC are available, and the typically low genomic levels of 5fC represent a particular sensitivity challenge. We here advance TALE-based nucleobase targeting from recognition to covalent cross-linking. We report TALE repeats bearing the ketone-amino acid p-acetylphenylalanine (pAcF) that universally bind all mammalian cytosine nucleobases, but selectively form diaminooxy-linker-mediated dioxime cross-links to 5fC. We identify repeat-linker combinations enabling single CpG resolution, and demonstrate the direct quantification of 5fC levels in a human genome background by covalent enrichment. This strategy provides a new avenue to expand the application scope of programmable probes with selectivity beyond A, G, T and C for epigenetic studies.

中文翻译:

用于直接捕获和定量 5-甲酰胞嘧啶的可编程蛋白质-DNA 交联

5-甲酰胞嘧啶 (5fC) 是哺乳动物基因组的表观遗传核碱基,作为活性 DNA 去甲基化的中间体出现。5fC 与关键核蛋白独特地相互作用和反应,表明基因组调控中的功能。转录激活因子样效应子 (TALE) 是基于重复的 DNA 结合蛋白,可用作直接、可编程识别和分析表观遗传核碱基的探针。然而,没有可用于选择性识别 5fC 的 TALE 重复序列,并且 5fC 的典型低基因组水平代表了一个特殊的敏感性挑战。我们在这里将基于 TALE 的核碱基靶向从识别推进到共价交联。我们报告了带有酮氨基酸对乙酰苯丙氨酸 (pAcF) 的 TALE 重复序列,其普遍结合所有哺乳动物胞嘧啶核碱基,但选择性地形成二氨基氧基接头介导的二肟交联至 5fC。我们确定了能够实现单个 CpG 分辨率的重复接头组合,并通过共价富集证明了人类基因组背景中 5fC 水平的直接量化。该策略提供了一种新的途径,可扩展可编程探针的应用范围,其选择性超出 A、G、T 和 C,用于表观遗传研究。
更新日期:2019-06-04
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