Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio-N⁶-isopentenyladenosine (ms²i⁶A), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. ms²i⁶A is traditionally identified by separating and digesting each tRNA from total RNA using RNA mass spectrometry. A transcriptome-wide and single-base resolution method that enables absolute mapping of ms²i⁶A along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach (redox activated chemical tagging sequencing, ReACT-seq) to detect ms²i⁶A transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, ms²i⁶A is bio-orthogonally tagged with an azide group without interference of canonical nucleotides, advancing enrichment of methylthio group modified RNAs prior to sequencing. ReACT-seq was demonstrated on nine known tRNAs and proved to be highly accurate, and the reverse transcription stop (RT-stop) character enables ReACT-seq detection at single-base resolution. In addition, ReACT-seq identified that the modification of ms²i⁶A is conservative and may not exist in other RNAs.

中文翻译：

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单碱基分辨率下 2-甲硫基-N6-异戊烯基腺苷的全转录组图谱

已鉴定出数百个修饰碱基并进行酶促修饰以转移 RNA (tRNA) 以调节各种生物体中的 RNA 功能。2-甲硫基-N ^{6 -}异戊烯基腺苷 (ms ² i ⁶ A) 是一种在 tRNA 第 37 位发现的超修饰碱基，存在于原核生物和真核生物中。ms ² i ⁶ A 传统上是通过使用 RNA 质谱法从总 RNA 中分离和消化每个 tRNA 来识别的。一种全转录组和单碱基分辨率方法，可实现 ms ² i ^{6的绝对映射}缺乏对其在不同 RNA 中分布的分析。在这里，通过化学选择性甲硫基生物偶联，我们引入了一种新方法（还原激活化学标记测序，ReACT-seq ）以单碱基分辨率检测全转录组 ms ² i ⁶ A。利用甲硫基和氧氮丙啶基团之间的化学选择性，ms ² i ⁶A 用叠氮基进行生物正交标记，不受规范核苷酸的干扰，从而在测序前促进甲硫基修饰的 RNA 的富集。ReACT-seq 在九个已知的 tRNA 上进行了演示，并被证明是高度准确的，逆转录终止（RT-stop）字符使 ReACT-seq 能够以单碱基分辨率进行检测。^{此外，ReACT-seq 发现 ms 2} i ⁶ A的修饰是保守的，可能不存在于其他 RNA 中。