Recent technological advances in the production of in vitro transcribed messenger RNA (IVT-mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT-mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT-mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT-mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell-free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification-dependent steps in the complex delivery process.

中文翻译：

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合成 mRNA 表达的反卷积：核苷化学改变可翻译性

体外转录信使 RNA (IVT-mRNA) 生产的最新技术进步促进了其临床应用及其在基础研究中的应用。在这方面，许多在内源性 mRNA 中自然观察不到的化学修饰主要是为了解决免疫原性问题并改善其生物学性能。然而，最近的研究结果表明，转染细胞中具有不同核苷修饰的 IVT-mRNA 的表达水平存在显着差异。鉴于 IVT-mRNA 递送和随后的细胞内表达的多步骤过程，尚不清楚哪个步骤受到 IVT-mRNA 化学作用的影响。在这里，我们对该过程进行了解卷积，并表明核苷修饰不会干扰载体的络合、其物理化学性质和细胞外稳定性，如所选修饰所示。mRNA 化学对核糖体蛋白质合成效率的直接影响是表达差异的一个因素，通过体外无细胞翻译进行量化。我们的结果表明，对于测试的核苷修饰，可翻译性是确定总体蛋白质产量的决定性步骤。对于未来合理选择定制合成 mRNA 化学物质的工作也特别重要，我们的研究结果设置了一个工作流程来识别复杂传递过程中潜在的限制性、依赖于修饰的步骤。