College of Chemistry and Molecular Sciences , Wuhan University , Wuhan , Hubei 430072 , China.
College of Life Science , Wuhan University , Wuhan , Hubei 430072 , China.
J Am Chem Soc. 2018 May 9;140(18):5886-5889. doi: 10.1021/jacs.7b13633. Epub 2018 Mar 5.
Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.
创新的检测技术可以实现哺乳动物转录组中 m6A 的精确分布，从而加深我们对其生物学功能的理解。我们特别引入了在脱氧胸苷三磷酸的 4 位用逐渐较大的原子（硫和硒）替代氧原子的方法，以削弱其与 m6A 碱基配对的能力，同时保持 A-T*碱基对与天然碱基对几乎相同。结果表明，4SedTTP 是一种出色的候选物，它赋予了 m6A 特定的 RT 截断特征，从而使得这种“RT 沉默”修饰可以在 m6A 去甲基酶 FTO 的协助下通过下一代测序来检测。这种不依赖抗体、涉及 4SedTTP 和 FTO 辅助的策略适用于 m6A 的识别，即使是在单核苷酸分辨率下，在未知区域中两个紧密聚集的 m6A 位点也是如此。
