m 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 m A sites is of utmost importance. However, m 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 m 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.

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 revealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA-and RNA-MTases, enabling sequence-specific modification ("writing") of plasmid DNA and light-triggered removal ("erasing").

Methyltransferases modify a wide range of biomolecules using S-adenosyl-l-methionine (AdoMet) as cosubstrate. Enzymatic generation of nucleoside-modified AdoMet analogues and conversion by different methyltransferases is shown.

Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo‐cleavable groups at methyltransferase target sites that block interactions with reader proteins until removed by non‐damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non‐natural groups, including first evidence for accepting photo‐cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC‐MjMAT) is 308‐fold more efficient at converting ortho‐nitrobenzyl‐(ONB)‐homocysteine than the wildtype enzyme. PC‐MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC‐MjMAT in complex with AdoONB and a red‐shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC‐MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC‐MjMAT exhibits remarkable activity on methionine analogues with red‐shifted ONB‐derivatives enabling photo‐deprotection of modified DNA by visible light.

Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on S-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic applications. We directly compared halide methyltransferase (HMT) and...