Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap Analog by In Vitro Transcription.
Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap Analog by In Vitro Transcription.
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DOI：
10.1002/cpz1.39
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发表时间：
2021-02-01
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影响因子：
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通讯作者：
Houston, Michael E
中科院分区：
文献类型：
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作者：
Henderson, Jordana M;Ujita, Andrew;Houston, Michael E
基于合成信使RNA（mRNA）的疗法是基因和细胞疗法、基因组工程、酶替代疗法以及现在在全球SARS-CoV-2大流行期间的疫苗开发的越来越受欢迎的方法。用于这种目的的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需要一个5 '-帽用于基因表达和mRNA稳定性。有两种方法在体外添加帽：通过两步多酶反应或共转录。与酶加帽相比，共转录方法使制备mRNA所需的反应步骤和酶最小化。CleanCap AG共转录加帽导致5 mg/ml的IVT具有94%的5 '-帽1结构。与第一代帽类似物（例如mCap和ARCA）相比，这是高效的，所述第一代帽类似物以较低的效率和反应产率并入帽0结构。本文描述了在IVT中使用TriLink Biotechnology的CleanCap AG进行共转录加帽。© 2021 Wiley Periodicals LLC.基本方案1：使用CleanCap的IVT基本方案2：mRNA纯化和分析。
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.