Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as ^m6A_m─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the ^m6A_m-cap-m⁷Gppp^Bn6A_mpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying ^Bn6A_m showed several advantages over typical capped transcripts. The ^Bn6A_m moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, ^Bn6A_m mRNAs provided higher protein yields than mRNAs carrying A_m or ^m6A_m, although the effect was cell-line-dependent. m⁷Gppp^Bn6A_mpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the ^Bn6A_m may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of ^m6A_m in mRNA.

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