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.

合成信使 RNA (mRNA) 是一种新兴的治疗平台，在肿瘤学和传染病领域具有重要应用。有效的 mRNA 药物必须由核糖体翻译，但不会引发强烈的核酸介导的免疫反应。为了扩展这些药物的药物化学工具箱，我们在此报告了合成 mRNA 中天然存在的核碱基 N ⁴ -乙酰胞苷 (ac4C) 的特性。我们发现 ac4C 与合成 mRNA 报告基因中的蛋白质生产兼容，但不会增强。然而，用 ac4C 替代胞苷可减少免疫细胞中由合成 mRNA 引起的炎症基因表达。化学蛋白质组捕获表明 ac4C 改变合成 mRNA 的蛋白质相互作用组，减少与胞苷结合蛋白和免疫传感器的结合。总体而言，我们的研究说明了 ac4C 调节 RNA-蛋白质相互作用的独特能力，并为使用 N ⁴ -胞苷酰化微调核酸治疗的特性奠定了基础。