New England Biolabs, Ipswich, Massachusetts 01938, USA.
RNA. 2022 Aug;28(8):1144-1155. doi: 10.1261/rna.079173.122. Epub 2022 Jun 9.
Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community.
mRNA 合成和脂质纳米粒技术的进步帮助实现了 mRNA 疗法和疫苗的应用。5' 帽结构是对合成 mRNA 进行功能化修饰的关键，以实现体内高效蛋白质翻译并逃避细胞固有免疫反应。5' 帽结构的掺入程度是 mRNA 生产的关键质量属性之一。RNA 帽分析涉及多个步骤：使用 RNase H、核酶或 DNA 酶从千碱基长的合成 mRNA 分子的 5' 端生成预定义的短片段，富集 5' 切割产物，并进行 LC-MS 完整质量分析。在本文中，我们描述了 (1) 使用 RNase H 进行位点特异性 RNA 切割的设计框架；(2) 一种用荧光标记 RNase H 切割片段的方法，以便使用更易于读取的方法，如凝胶电泳或高通量毛细管电泳；(3) 一种在 RNase H 后使用带有去硫生物素的寡核苷酸和链霉亲和素磁珠进行简化的纯化方法，然后使用水进行洗脱。通过提供一种基于 RNase H 的 RNA 5' 帽分析的设计框架，使用资源密集度较低的分析方法，我们希望使 RNA 帽分析更容易为科学界所接受。
