Trinucleotide mRNA
Cap Analogue N6‑Benzylated at the Site of
Posttranscriptional m6Am Mark Facilitates mRNA
Purification and Confers Superior
Translational Properties In Vitro and In Vivo
posted on 2024-03-05, 17:40authored byMarcin Warminski, Edyta Trepkowska, Miroslaw Smietanski, Pawel J. Sikorski, Marek R. Baranowski, Marcelina Bednarczyk, Hanna Kedzierska, Bartosz Majewski, Adam Mamot, Diana Papiernik, Agnieszka Popielec, Remigiusz A. Serwa, Brittany A. Shimanski, Piotr Sklepkiewicz, Marta Sklucka, Olga Sokolowska, Tomasz Spiewla, Diana Toczydlowska-Socha, Zofia Warminska, Karol Wolosewicz, Joanna Zuberek, Jeffrey S. Mugridge, Dominika Nowis, Jakub Golab, Jacek Jemielity, Joanna Kowalska
Eukaryotic mRNAs undergo cotranscriptional 5′-end
modification
with a 7-methylguanosine cap. In higher eukaryotes, the cap carries
additional methylations, such as m6Ama
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 m6Am-cap–m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped
transcripts. The Bn6Am 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, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, 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 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 Bn6Am may pave the way for more potent mRNA-based vaccines
and therapeutics and serve as molecular tools to unravel the role
of m6Am in mRNA.