PDF(Pubmed)
Abstract：
The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is relatively inefficient, resulting in <20% C:T mismatches at a fully modified ac4C site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for comprehensive mapping. Here, we introduce \"RetraC:T\" (reduction to tetrahydro-ac4C and reverse transcription with amino-dATP to induce C:T mismatches) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent sodium cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to tetrahydro-ac4C. Incorporation of the modified dNTP substantially improved C:T mismatch rates, reaching stoichiometric detection of ac4C in 18S rRNA. Importantly, 2-amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting ac4C at base resolution.
摘要：
RNA中表位基因组修饰的功能分析受到缺乏准确捕获其位置和水平的方法的限制。我们先前证明，RNA修饰N4-乙酰胞苷（ac4C）可以通过硼氢化钠还原为四氢乙酰胞苷（四氢-ac4C）以碱基分辨率定位，随后进行cDNA合成，使腺苷错误掺入相对于减少的ac4C位点，在乙酰化的胞苷(RedaC:T)处达到C:T错配。然而,这个过程相对低效，在18SrRNA中的完全修饰的ac4C位点处导致小于20%的C:T错配。考虑到包括mRNA在内的其他底物中的ac4C位置不太可能达到完全外显率，这种方法对于全面映射并不理想。这里,我们引入了“RetraC：T”（还原为四氢ac4C并用氨基dATP进行逆转录以诱导C：T错配）作为一种增强检测细胞RNA中ac4C的能力的方法。简而言之,RNA通过NaBH4或密切相关的试剂氰基硼氢化钠（NaCNBH3）还原，然后在修饰的DNA核苷酸存在下进行cDNA合成，2-氨基-dATP,优先结合四氢-ac4C。引入改良的dNTP大大提高了C：T失配率，在18SrRNA中达到ac4C的化学计量检测。重要的是,2-氨基-dATP不会导致截短的cDNA产物，也不会增加其他位置的错配。因此，修改后的dNTP作为新添加到工具箱中，用于以基本分辨率检测ac4C。
