合成信息 RNA (mRNA) 的治疗用途已在 COVID-19 疫苗中得到验证，并在开发传染性和肿瘤疫苗方面显示出巨大潜力。然而，体外转录 (IVT) 过程中产生的双链 RNA (dsRNA) 副产物会降低基于 mRNA 的疗法的功效并引发先天免疫反应。现有消除 dsRNA 副产物的方法通常很麻烦且劳动密集型。在这项研究中，我们发现松散的 mRNA 二级结构和序列中更多不配对的 U 碱基通常会导致 IVT 过程中形成更多的 dsRNA 副产物。我们进一步开发了基于序列特征的 dsRNA 副产物形成的预测模型，以指导 mRNA 序列的优化，有助于最大限度地减少不需要的免疫反应并提高 mRNA 产物的蛋白质表达。总的来说，我们的研究为开发有效的 mRNA 疗法提供了新的线索和方法，最大限度地减少 dsRNA 副产物并增加蛋白质表达。版权所有 © 2024。由 Elsevier Ltd 出版。

The therapeutic use of synthetic message RNA (mRNA) has been validated in COVID-19 vaccines and shows enormous potential in developing infectious and oncological vaccines. However, double-stranded RNA (dsRNA) byproducts generated during the in vitro transcription (IVT) process can diminish the efficacy of mRNA-based therapeutics and provoke innate immune responses. Existing methods to eliminate dsRNA byproducts are often cumbersome and labor-intensive. In this study, we revealed that a loose mRNA secondary structure and more unpaired U bases in the sequence generally lead to the formation of more dsRNA byproducts during the IVT process. We further developed a predictive model for dsRNA byproducts formation based on sequence characteristics to guide the optimization of mRNA sequences, helping to minimize unwanted immune response and improve the protein expression of mRNA products. Collectively, our study provides novel clues and methodologies for developing effective mRNA therapeutics with minimized dsRNA byproducts and increased protein expression.Copyright © 2024. Published by Elsevier Ltd.