Enzymatic or In Vivo Installation of Propargyl Groups in Combination with Click Chemistry for the Enrichment and Detection of Methyltransferase Target Sites in RNA

m⁶ A 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 m⁶ A sites is of utmost importance. However, m⁶ A 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 groups can be introduced enzymatically by wild-type METTL3-METTL14. Reverse transcription terminated up to 65 % at m⁶ A 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.