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

Eukaryotic mRNAs are modified at their 5′-end by the addition of a 7-methylguanosine cap connected to the first transcribed nucleotide by a triphosphate chain. In higher eukaryotes, the cap carries additional marks in the form of 2′-O-methyl groups at the first or the first and the second transcribed nucleotide. If adenosine initiates the transcript, it is often additionally methylated at the N6-position to form m6Am - an epitranscriptomic mark unique for mRNA 5′-end. Here, we designed and synthesized an N6-benzyl analog of m6Am-Bn6Am - and incorporated it into mRNA 5′-end using a novel trinucleotide cap analog (m7GpppBn6AmpG, AvantCap) and T7 polymerase. Capped mRNAs carrying Bn6Am have several advantages over typically capped mRNAs. The Bn6Am moiety works as an mRNA purification handle, enabling the separation of capped RNA from residual uncapped RNA species. mRNAs produced with AvantCap contain fewer dsRNA than reference caps. In cultured cells, Bn6Am mRNAs can provide higher protein outputs than mRNAs carrying Am or m6Am, although the effect is cell line-dependent. Finally, m7GpppBn6AmpG-capped mRNAs encoding reporter proteins intravenously delivered into mice provide up to 6-fold higher protein outputs than reference mRNAs. m7GpppBn6AmpG-capped mRNAs encoding tumor antigens show superior activity in therapeutic settings such as anti-cancer vaccines. AvantCap is recognized by eIF4E in vitro similarly to physiological caps, but in a pull-down assay with cell extracts, the protein is enriched for Bn6Am compared to Am cap. eIF3 complex, involved in alternative translation initiation, is also enriched for AvantCap. The m7GpppBn6AmpG-capped mRNAs are efficiently decapped by Dcp2 but, but are not susceptible to dealkylation by RNA demethylase FTO. The biochemical characterization suggests several phenomena underlying the biological properties: (i) increasing competitiveness of mRNA 5′-end by decreasing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or combination of these effects. AvantCapped-mRNAs carrying Bn6Am mark may pave the way to more potent mRNA-based vaccines and therapeutics and serve as molecular tools for deciphering the role of the m6Am mark in mRNA. ### Competing Interest Statement J.J., J.K., P.J.S., and M.W. are inventors of a patent related to AvantCap. Some of the authors are shareholders of Explorna Therapuetics.