Synthetic mRNA therapeutics offer a versatile platform for treating diverse conditions, including cancer and infectious diseases. For delivery into cells, these mRNAs are encapsulated in lipid nanoparticles and commonly incorporate modified ribonucleotides to improve stability, enhance translation and mitigate immune recognition1. N1-Methylpseudouridine (m1Ψ) has become the industry standard for synthetic mRNAs owing to its effectiveness in promoting translation and reducing immunogenicity2. However, recent studies have shown that m1Ψ can compromise translational fidelity, leading to errors such as premature termination and ribosomal frameshifting3–5. Here we reveal N4-acetylcytidine (ac4C) as a functionally distinct alternative to m1Ψ. Across cultured cell lines, primary human monocyte-derived dendritic cells and mouse liver, ac4C suppressed inflammatory responses as effectively as m1Ψ while driving higher protein yields. Single-molecule imaging of translation revealed broadly similar ribosome densities per mRNA for ac4C-modified and m1Ψ-modified transcripts. However, translation elongation with m1Ψ-modified mRNA was nearly twofold slower than with ac4C, which resulted in reduced protein output and increased ribosome collisions that further limited protein production through the engagement of quality-control pathways and +1 frameshifting. These findings underscore the importance of context in designing therapeutic mRNAs and position the translation elongation rate as a key determinant of the efficacy of modified ribonucleotides.
