Department of Chemistry and Chemical Biology, Technical University of Dortmund , Otto-Hahn-Str. 6, 44227 Dortmund, Germany.
J Am Chem Soc. 2016 Aug 10;138(31):9910-8. doi: 10.1021/jacs.6b04807. Epub 2016 Aug 1.
We report the direct isolation of user-defined DNA sequences from the human genome with programmable selectivity for both canonical and epigenetic nucleobases. This is enabled by the use of engineered transcription-activator-like effectors (TALEs) as DNA major groove-binding probes in affinity enrichment. The approach provides the direct quantification of 5-methylcytosine (5mC) levels at single genomic nucleotide positions in a strand-specific manner. We demonstrate the simple, multiplexed typing of a variety of epigenetic cancer biomarker 5mC with custom TALE mixes. Compared to antibodies as the most widely used affinity probes for 5mC analysis, i.e., employed in the methylated DNA immunoprecipitation (MeDIP) protocol, TALEs provide superior sensitivity, resolution and technical ease. We engineer a range of size-reduced TALE repeats and establish full selectivity profiles for their binding to all five human cytosine nucleobases. These provide insights into their nucleobase recognition mechanisms and reveal the ability of TALEs to isolate genomic target sequences with selectivity for single 5-hydroxymethylcytosine and, in combination with sodium borohydride reduction, single 5-formylcytosine nucleobases.
我们报告了一种从人类基因组中直接分离用户定义的 DNA 序列的方法，该方法对经典和表观遗传核碱基具有可编程选择性。这是通过使用工程转录激活子样效应物（TALEs）作为 DNA 大沟结合探针在亲和富集中实现的。该方法提供了在单链特异性方式下直接定量单个基因组核苷酸位置的 5-甲基胞嘧啶（5mC）水平的方法。我们展示了使用定制 TALE 混合物对多种表观遗传癌症生物标志物 5mC 的简单、多重分型。与作为最广泛用于 5mC 分析的亲和力探针的抗体相比，即用于甲基化 DNA 免疫沉淀（MeDIP）方案的抗体，TALEs 提供了更高的灵敏度、分辨率和技术易用性。我们设计了一系列尺寸减小的 TALE 重复序列，并建立了它们与所有五种人类胞嘧啶核碱基结合的完全选择性图谱。这些为它们的核碱基识别机制提供了深入了解，并揭示了 TALEs 具有选择性地分离基因组靶序列的能力，对于单个 5-羟甲基胞嘧啶，并且与硼氢化钠还原结合，单个 5-甲酰胞嘧啶核碱基。
