Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional^m6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

Abstract

Eukaryotic mRNAs undergo co-transcriptional 5’-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such asm6Am– 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-resistantN6-benzyl analog of them6Am-cap – m7GpppBn6AmpG (termedAvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carryingBn6Amshowed several advantages over typical capped transcripts. TheBn6Ammoiety 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,Bn6AmmRNAs provided higher protein yields than mRNAs carrying Amorm6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-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 ofAvantCap: (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 theBn6Ammay pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of them6Amin mRNA.