RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

中文翻译：

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通过二级结构设计稳定信使RNA的理论基础

RNA 水解给基于信使 RNA (mRNA) 的疫苗和治疗药物的制造、长期储存、全球递送和体内稳定性带来了问题。减少 mRNA 水解的一种很大程度上尚未探索的策略是重新设计 RNA 以形成双链区域，这些区域在编码相同蛋白质的同时免受内联切割和酶促降解。这种策略可以提供的稳定程度以及实现稳定的最有效算法方法仍然知之甚少。在这里，我们提出了用于估计 RNA 水解稳定性的简单计算，以及将 mRNA 的平均不配对概率（AUP）与其总体水解速率联系起来的模型。为了表征通过结构设计可实现的稳定性，我们将传统 mRNA 设计方法的 AUP 优化与通过 Eterna 平台上的 OpenVaccine 挑战获得的计算更复杂的算法和众包的结果进行了比较。我们发现 Eterna 上的合理设计和更复杂的算法导致构建具有低 AUP 的结构，我们将其称为“超级文件夹”mRNA。这些设计表现出广泛多样性的序列和结构特征，这些特征对于翻译、生物物理大小和免疫原性来说可能是理想的。此外，它们的折叠对温度、计算机建模方法、侧翼非翻译区的选择以及靶蛋白序列的变化具有鲁棒性，如 B.1.351、P.1 和 B.1.1.7 变体的超级文件夹 mRNA 的快速重新设计所示。灌注前稳定的 SARS-CoV-2 刺突蛋白。体外 mRNA 半衰期至少增加两倍似乎可以立即实现。