Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA

Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here we report the properties of the naturally occurring nucleobase N⁴-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, our studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N⁴-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.