Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional m6 Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

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Eukaryotic mRNAs undergo co-transcriptional 5’-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6 A 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 N 6-benzyl analog of the m6 A m -cap – m 7 Gppp Bn6 A m pG (termed AvantCap ) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6 A m showed several advantages over typical capped transcripts. The Bn6 A m moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6 A m mRNAs provided higher protein yields than mRNAs carrying A m or m6 A m , although the effect was cell line-dependent. m 7 Gppp Bn6 A m pG-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 setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap : (i) increased competitiveness of the mRNA 5’-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped- mRNAs bearing the Bn6 A m may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6 A m in mRNA.