Nature communicationsSep 28, 2026

Using N1-methylpseudouridine changes to improve mRNA vaccine design

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Sep 30, 2026

Abstract

m¹Ψ increases +1 frameshifting on UUUC motifs in therapeutic mRNAs.

The modification m¹Ψ is associated with increased ribosomal frameshifting, which can generate aberrant peptides.
Frameshifting occurs due to peptidyl-tRNA pausing in the ribosomal P site.
m¹Ψ weakens codon-anticodon interactions and promotes a tRNA conformation conducive to frameshifting.
Changing UUUC motifs to UUCC or UUUU eliminates the frameshifting effect.
Codon optimization may mitigate the risks associated with m¹Ψ in therapeutic mRNA design.

Simplified

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.