A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Abstract

Methyltransferases have proven useful to install functional groups site-specifically in different classes of biomolecules when analogues of their cosubstrate S-adenosyl-l-methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site-specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell-permeable, thus preventing implementation of this strategy in cells. We present a two-step enzymatic cascade for site-specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5' cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.

Dual 5′ Cap Labeling Based on Regioselective RNA Methyltransferases and Bioorthogonal Reactions

“…AdoMet analogues are chemically synthesized from S ‐adenosylhomocysteine, but they can be prepared enzymatically from methionine analogues, which mimic the biosynthesis of AdoMet . To analyze whether a straightforward double modification of compound 1 was also possible from methionine analogues in one pot, we established a three‐step cascade reaction.…”

Enzymatischer oder In‐vivo‐Einbau von Propargylgruppen in Kombination mit Klick‐Chemie zur Anreicherung und Detektion von Methyltransferase‐Zielsequenzen in RNA

“…[19] HeLa-Zellen wurden mit Propargyl-l-selenohomocystein (2)behandelt, die Total-RNAwurde isoliert und die Bildung von 2'-O-Propargyladenosin mittels LC-MS/MS nachgewiesen (Abbildungen 3A und S12). Zu diesem Zweck haben wir eine Strategie zur metabolischen Markierung dieser Sequenzen mithilfe der Biosynthese von AdoMet-Analoga aus Methionin-Analoga und ATPd urch Methionin-Adenosyl-Transferase (MAT) entwickelt (Schema 1B).…”

The notorious R.N.A. in the spotlight - drug or target for the treatment of disease

“…There is no need to restrict ourselves to the usage of nucleases, like RISC, RNaseH, and RNaseP 232 . Just to give a few examples, there are RNA editing and modifying enzymes inside the cell that can change nucleotides (A-to-I, C-to-U 233 , U-to-ψ, A-to-m6A, 234 and many more for the tRNAs 235 ), that add the cap 236 and the poly(A)-tail, 237 RNAs can be precisely processed, for instance by the CCA-adding enzymes, 238,239 TUTases, 240 etc 241 . Thus, even complex repair processes are conceivable, including the repair of insertion and deletion mutations at the RNA-level.…”