Recoding by N1-methylpseudouridine guides rational mRNA vaccine design

Abstract N 1 -methylpseudouridine (m 1 Ψ) is a key modification used in SARS-CoV-2 mRNA vaccines that reduces immunogenicity and increases mRNA stability. Recent studies suggested that m 1 Ψ can promote ribosomal frameshifting, a translational error generating aberrant peptides that elicit immune responses, raising concerns about unintended antigenicity. Here, we systematically examined the efficiency of frameshifting induced by m 1 Ψ and the underlying mechanism, aiming to inform future mRNA vaccine design. Using mRNA-based dual-fluorescence reporters in cells, fully in vitro reconstituted translation system, and single-molecule FRET microscopy, we show that m 1 Ψ increases +1 frameshifting on UUUC motifs in therapeutic mRNAs. Frameshifting occurs when the peptidyl-tRNA pauses in the ribosomal P site, where m 1 Ψ both weakens codon-anticodon interactions and promotes a frameshifting-prone tRNA conformation. Replacing slippery UUUC motifs with UUCC or UUUU eliminates this effect. Our results reveal how a clinically relevant mRNA modification promotes recoding and show that codon optimization mitigates this risk in therapeutic mRNA design.