Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
Department of Biochemistry, Stanford University, Stanford, CA, 94305, USA.
Nat Commun. 2022 Mar 22;13(1):1536. doi: 10.1038/s41467-022-28776-w.
Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop an RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that highly structured "superfolder" mRNAs can be designed to improve both stability and expression with further enhancement through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.
治疗性 mRNA 和疫苗正在被开发用于广泛的人类疾病，包括 COVID-19。然而，它们的优化受到 mRNA 不稳定性和蛋白质表达效率低下的阻碍。在这里，我们描述了克服这些障碍的设计原则。我们开发了一种称为 PERSIST-seq 的 RNA 测序为基础的平台，用于系统地描绘细胞内 mRNA 的稳定性、核糖体负载以及多样化 mRNA 库的溶液稳定性。我们发现，令人惊讶的是，细胞内稳定性是蛋白质产量的一个更大的驱动因素，而不是高核糖体负载。我们进一步引入了一种称为 In-line-seq 的方法，应用于数千种不同的 RNA，揭示了减轻水解降解的基于序列和结构的规则。我们的研究结果表明，可以设计高度结构化的“超级折叠”mRNA，以提高稳定性和表达，进一步通过假尿嘧啶核苷修饰进行增强。总之，我们的研究表明，同时提高了 mRNA 的稳定性和蛋白质的表达，并为 mRNA 药物的增强提供了一个计算实验平台。
