Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics
Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics
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DOI：
10.1101/2021.03.29.437587
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发表时间：
2022-03-22
影响因子：
16.6
通讯作者：
Das, Rhiju
中科院分区：
文献类型：
--
作者：
Leppek, Kathrin;Byeon, Gun Woo;Das, Rhiju
作者开发了一个基于RNA测序的平台，persistence -seq，以同时描述细胞内mRNA稳定性，核糖体负载和不同mRNA文库的溶液稳定性，从而得出改进mRNA治疗的设计原则。针对包括COVID-19在内的多种人类疾病的治疗性mrna和疫苗正在开发中。然而，它们的优化受到mRNA不稳定和低效率蛋白表达的阻碍。在这里，我们将描述克服这些障碍的设计原则。我们开发了一个基于RNA测序的平台，称为persistence -seq，以系统地描述细胞内mRNA稳定性，核糖体负载以及各种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输出的更大驱动因素，而不是高核糖体负载。我们进一步介绍了一种称为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，高度结构化的“超级文件夹”mrna可以通过假尿嘧啶核苷修饰进一步提高稳定性和表达。总之，我们的研究证明了mRNA稳定性和蛋白质表达的同时改善，为mRNA药物的增强提供了一个计算实验平台。
The authors develop an RNA sequencing-based platform, PERSIST-seq, to simultaneously delineate in-cell mRNA stability, ribosome load, and in-solution stability of a diverse mRNA library to derive design principles for improved mRNA therapeutics.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.