ac4C does not impair in vitro transcription with T7 polymerase

These mRNAs are delivered to cells encapsulated in lipid nanoparticles (LNPs) and commonly incorporate modified ribonucleotides to improve their stability and translation while mitigating the immune response to foreign RNA1. N1-methylpseudouridine (m1Ψ) has become the industry standard for synthetic mRNAs due to its effectiveness in enhancing translation and reducing immunogenicity2.

However, recent studies have shown that m1Ψ can compromise translational fidelity, leading to errors such as ribosomal frameshifting3,4. Here, we reveal N4-acetylcytidine (ac4C) as a promising alternative to m1Ψ for use in therapeutic mRNAs. In comparing synthetic mRNA translation in cell lines, primary human monocyte-derived dendritic cells and murine liver, we found that while both ac4C and m1Ψ suppress the inflammatory response to synthetic mRNA, ac4C consistently results in higher protein yields.