The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.
© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.