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Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5′ cap is important for mRNA translation and immune response and can be naturally methylated at different positions by  S ‐adenosyl‐ l ‐methionine (AdoMet)‐dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the  N  6 ‐position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo‐enzymatic propargylation enabled production of site‐specifically modified reporter‐mRNAs. These cap‐propargylated mRNAs were efficiently translated and showed ≈3‐fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS‐CoV‐2—a currently tested epitope for mRNA vaccination—was used. Site‐specific chemo‐enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.

Chemo‐Enzymatic Modification of the 5′ Cap Maintains Translation and Increases Immunogenic Properties of mRNA