Recoding by N1-methylpseudouridine guides rational mRNA vaccine design.

N1-methylpseudouridine (m1Ψ) is a key modification used in SARS-CoV-2 mRNA vaccines that reduces immunogenicity and increases mRNA stability. Recent studies suggested that m1Ψ 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 m1Ψ 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 m1Ψ increases +1 frameshifting on UUUC motifs in therapeutic mRNAs. Frameshifting occurs when the peptidyl-tRNA pauses in the ribosomal P site, where m1Ψ 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.