RNA labelling has become indispensable in studying RNA biology. Nucleoside analogues with a chemical sequencing power represent desirable RNA labelling molecules because precise labelling information at base resolution can be obtained. Here, we report a new nucleoside analogue, N⁴-allylcytidine (a⁴C), which is able to tag RNA through both in vitro and in vivo pathways and further specifically reacts with iodine to form 3, N⁴-cyclized cytidine (cyc-C) in a catalyst-free, fast and complete manner. Full spectroscopic characterization concluded that cyc-C consisted of paired diastereoisomers with opposite chiral carbon centers in the fused 3, N⁴-five-membered ring. During RNA reverse transcription into complementary DNA, cyc-C induces base misincorporation due to the disruption of canonical hydrogen bonding by the cyclized structure and thus can be accurately identified by sequencing at single base resolution. With the chemical sequencing rationale of a⁴C, successful applications have been performed including pinpointing N⁴-methylcytidine methyltransferases’ substrate modification sites, metabolically labelling mammalian cellular RNAs, and mapping active cellular RNA polymerase locations with the chromatin run-on RNA sequencing technique. Collectively, our work demonstrates that a⁴C is a promising molecule for RNA labelling and chemical sequencing and expands the toolkit for studying sophisticated RNA biology.

中文翻译：

---

N 4-烯丙基胞苷：一种用于 RNA 标记和化学测序的新型核苷类似物

RNA标记已成为研究RNA生物学不可或缺的一部分。具有化学测序能力的核苷类似物代表了理想的RNA标记分子，因为可以获得碱基分辨率的精确标记信息。在这里，我们报道了一种新的核苷类似物N ⁴ -烯丙基胞苷（a ⁴ C），它能够通过体外和体内途径标记RNA，并进一步与碘特异性反应形成3, N ^{4 -}环化胞苷（cyc -C) 以无催化剂、快速且完整的方式。完整的光谱表征得出结论，cyc-C 由在稠合 3, N ⁴ -五元环中具有相反手性碳中心的成对非对映异构体组成。在RNA逆转录成互补DNA的过程中，cyc-C由于环化结构破坏了规范氢键而诱导碱基错误掺入，因此可以通过单碱基分辨率的测序来准确识别。^凭借4 C的化学测序原理，已经进行了成功的应用，包括精确定位N ⁴ -甲基胞苷甲基转移酶的底物修饰位点、代谢标记哺乳动物细胞 RNA 以及使用染色质连续 RNA 测序技术绘制活性细胞 RNA 聚合酶位置。总的来说，我们的工作表明⁴ C 是一种很有前途的 RNA 标记和化学测序分子，并扩展了研究复杂 RNA 生物学的工具包。