Enzymatic or in vivo installation of propargyl groups in combination with click chemistry enables enrichment and detection of methyltransferase target sites in RNA

m6A is the most abundant internal modification in eukaryotic mRNA. It is introduced by METTL3-METTL14 and tunes mRNA metabolism, impacting cell differentiation and development. Precise transcriptome-wide assignment of m6A sites is of utmost importance. However, m6A does not interfere with Watson-Crick base pairing making polymerase-based detection challenging. We developed a chemical biology approach for the precise mapping of methyltransferase (MTase) target sites based on the introduction of a bioorthogonal propargyl group in vitro and in cells. We show that propargyl can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m6A sites after bioconjugation and purification, hence enabling detection of METTL3-METTL14 target sites by next generation sequencing. Importantly, we implemented metabolic propargyl labeling of RNA MTase target sites in vivo based on propargyl-L-selenohomocysteine and validated different types of known rRNA methylation sites. Overall design: enrichment of methylated nucleotides by bioconjugation of propagyl groups in synthetic oligos and rRNAm6A是真核生物信使RNA（messenger RNA, mRNA）中含量最为丰富的内部转录后修饰。该修饰由METTL3-METTL14复合物催化引入，可调控mRNA代谢过程，进而影响细胞分化与个体发育。在全转录组范围内精准定位m6A修饰位点具有极高的研究价值与重要意义。然而，m6A修饰并不干扰沃森-克里克碱基配对（Watson-Crick base pairing），这使得基于聚合酶的检测技术面临较大挑战。我们开发了一种化学生物学策略，通过在体外与活细胞内引入生物正交炔丙基（propargyl）基团，实现甲基转移酶（methyltransferase, MTase）靶标位点的精准图谱绘制。实验证实，野生型METTL3-METTL14可通过酶促反应引入炔丙基基团。在完成生物偶联与纯化步骤后，逆转录反应在m6A修饰位点处的终止效率可达65%，借此可通过下一代测序（next generation sequencing, NGS）技术检测METTL3-METTL14的靶标修饰位点。尤为重要的是，我们基于炔丙基-L-硒代高半胱氨酸（propargyl-L-selenohomocysteine），实现了活细胞内RNA甲基转移酶靶标位点的代谢炔丙基标记，并验证了多种已报道的核糖体RNA（ribosomal RNA, rRNA）甲基化位点。整体实验设计：通过对合成寡核苷酸与rRNA中的炔丙基基团进行生物偶联，实现甲基化核苷酸的富集。