Tag‐Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases

Abstract

The mRNA modification N6‐methyladenosine (m6A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3‐METTL14 and METTL16 act as “writers” for different target transcripts and sequence motifs. The modification is perceived by dedicated “reader” and “eraser” proteins, but not by polymerases. We report that METTL3‐14 shows remarkable cosubstrate promiscuity, enabling sequence‐specific internal labeling of RNA without additional guide RNAs. The transfer of ortho‐nitrobenzyl and 6‐nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase‐catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo‐enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo‐enzymatic modification of different sites in a single RNA.

Biomimetic S‐Adenosylmethionine Regeneration Starting from Multiple Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes**

“…Besides SAH, MTAN cleaves the SAM‐derived byproducts DOA (from radical SAM enzymes) and MTA (from ACP transfers and polyamine biosynthesis), yielding the corresponding ribose derivative and adenine [40,50] . SAM supply cascades are well established and have been used for many different scenarios including conventional SAM‐dependent MTs and radical SAM enzymes [33,34,47,51–60] . Nevertheless, depending on the application, a cofactor regeneration system is desired that does not require stoichiometric amounts of the precursors ATP or CldA.…”

DNA Tiling Enables Precise Acylation‐Based Labeling and Control of mRNA

“…One recent development involves ribozyme‐assisted labeling by selectively catalyzing the reaction of a targeted adenosine with N 6 ‐modified ATP or non‐natural nucleotide analogues; [14] while elegant, the approach is limited to adenosines and cannot be reversed. Another method utilizes promiscuous mRNA methyltransferases and their modified substrates for tag‐free internal RNA labeling and caging, but is limited to specific consensus sequences [15] . Classical RNA‐reactive chemistries tend to exhibit low yields and usually cannot be programmed to specific sites; examples include photocrosslinking with psoralens, which are selective only for double‐stranded regions, [16] dimethylsulfate, which generally reacts at unpaired cytosines and adenines, [17] and stochastic reaction with 2′‐OH acylating reagents that form bonds at 2′‐OH groups of unpaired nucleotides [18] .…”