ac4C mRNA Modification Delivers Higher Protein Output With Maintained Fidelity
A Nature study shows N4-acetylcytidine (ac4C) in synthetic IVT mRNA yields higher protein output than N1-methylpseudouridine (m1Ψ) across cells and in mice, while suppressing innate immune activation to a similar extent as m1Ψ. ac4C slows elongation modestly but avoids the ribosome collisions and +1 frameshifting that m1Ψ promotes, boosting translation efficiency without compromising fidelity. In primary human immune cells, ac4C reduces IFN responses while increasing NanoLuc output; in mice, ac4C mRNA–LNPs produced ~3.5-fold higher hepatic luminescence at 24 h than m1Ψ. The work, using SINAPs imaging, ribosome profiling and in vivo models, supports a BUMP model where different nucleotide modifications steer ribosome flow and quality control. While m1Ψ remains a strong clinical standard, ac4C offers a complementary option for contexts demanding high yield and accurate protein production.