2'-O-Methyltransferase is expressed in Escherichia coli. Using S-adenosylmethionine (SAM) as the methyl donor, this enzyme adds a methyl group to the 2'-O position of the first nucleotide immediately adjacent to the Cap0 structure at the 5'-end of RNA, forming the Cap1 structure. The enzyme only uses RNA bearing the 7-methylguanosine cap structure (m7GpppN) as substrate and does not act on RNA with 5'-terminal pN, ppN, pppN or GpppN. The Cap1 structure enhances mRNA translation efficiency and improves expression during mRNA transfection, making it an essential modification for in vitro mRNA synthesis.

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