Department of Molecular Biophysics & Biochemistry , Yale University , New Haven , Connecticut 06511 , United States.
Chemical Biology Institute , Yale University , West Haven , Connecticut 06516 , United States.
J Am Chem Soc. 2018 Nov 7;140(44):14567-14570. doi: 10.1021/jacs.8b08554. Epub 2018 Oct 24.
RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (sU), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (sG). Using TimeLapse sequencing (TimeLapse-seq), sG can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of sG recoding experiments to reveal transcriptome-wide RNA population dynamics.
RNA 测序（RNA-seq）可测量生物样本中 RNA 的丰度，但无法提供有关测序 RNA 的时间信息。代谢标记可用于区分新合成的 RNA 和预先存在的 RNA。通过对代谢标记的氢键模式进行化学重编码诱导的突变，可以揭示在测序实验背景下哪些 RNA 是新的。这些核苷酸重编码策略已针对单个尿嘧啶类似物 4-硫代尿嘧啶（sU）进行了开发，从而限制了这些实验的范围。在这里，我们报告了首次使用鸟苷类似物 6-硫代鸟嘌呤（sG）进行核苷重编码。使用时程序列（TimeLapse-seq），sG 可以在 RNA 友好的氧化亲核-芳香取代条件下被重编码为腺嘌呤类似物（取代的 2-氨基腺嘌呤）。我们展示了首次使用 sG 重编码实验来揭示转录组范围的 RNA 群体动态。
