College of Chemistry and Molecular Sciences, Department of Hematology of Zhongnan Hospital, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, Hubei 430072, People's Republic of China.
State Key Laboratory of Virology and Medical Research Institute, Hubei Province Key Laboratory of Allergy and Immunology and Department of Immunology, Wuhan University School of Medicine, Wuhan 430071, People's Republic of China.
J Am Chem Soc. 2023 Mar 8;145(9):5467-5473. doi: 10.1021/jacs.2c13894. Epub 2023 Feb 23.
Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio--isopentenyladenosine (msiA), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. msiA 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 msiA along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach (dox ctivated hemical agging uencing, ReACT-seq) to detect msiA transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, msiA 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 msiA is conservative and may not exist in other RNAs.
已经鉴定并酶修饰了数百种碱基，以将转移 RNA（tRNA）转移到各种生物体中，从而调节 RNA 功能。2-甲基硫代-异戊烯基腺苷（msiA）是一种在 tRNA 位置 37 处发现的超修饰碱基，存在于原核生物和真核生物中。msiA 传统上是通过使用 RNA 质谱法从总 RNA 中分离和消化每个 tRNA 来鉴定的。缺乏一种能够在单碱基分辨率上绝对绘制 msiA 图谱并分析其在不同 RNA 中分布的全转录组和单碱基分辨率方法。在这里，通过化学选择性硫甲基化生物缀合，我们引入了一种新方法（dox 激活化学标记测序，ReACT-seq），以在单碱基分辨率上全转录组检测 msiA。利用甲基硫基团和恶唑啉基团之间的化学选择性，msiA 可以与叠氮基团进行生物正交标记，而不会干扰规范核苷酸，从而在测序前优先富集甲基硫修饰的 RNA。ReACT-seq 在 9 个已知的 tRNA 上进行了验证，结果证明其高度准确，并且逆转录停止（RT-stop）特征使其能够在单碱基分辨率下进行检测。此外，ReACT-seq 还鉴定出 msiA 的修饰是保守的，可能不存在于其他 RNA 中。
