Trinucleotide mRNA Cap Analogue N 6-Benzylated at the Site of Posttranscriptional m6 A m Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 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 analogue 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 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, 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 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 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 m6 A m in mRNA.