TriLink Biotechnologies LLC, San Diego, California.
Curr Protoc. 2021 Feb;1(2):e39. doi: 10.1002/cpz1.39.
Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.
基于合成信使 RNA（mRNA）的疗法是一种越来越受欢迎的基因和细胞治疗、基因组工程、酶替代疗法的方法，现在在全球 SARS-CoV-2 大流行期间，mRNA 还被用于疫苗开发。出于此类目的的 mRNA 可以通过酶促体外转录（IVT）反应合成，并配制用于体内递送。成熟的 mRNA 需要 5' - 帽来进行基因表达和 mRNA 稳定性。有两种方法可以在体外添加帽：通过两步多酶反应或共转录。与酶加帽相比，共转录方法可最大程度减少合成 mRNA 所需的反应步骤和酶。CleanCap AG 共转录加帽可在 IVT 中获得 5mg/ml 的产量，其中 94% 为 5' - 帽 1 结构。与第一代帽类似物（如 mCap 和 ARCA）相比，这是非常高效的，因为前者以较低的效率和反应产率掺入帽 0 结构。本文描述了使用 TriLink Biotechnology 的 CleanCap AG 在 IVT 中进行共转录加帽。© 2021 威立出版公司。基础方案 1：使用 CleanCap 的 IVT 基础方案 2：mRNA 纯化和分析。
