Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription

• Main Authors: Cabral, C.R (Author), Escamilla-Powers, J.R (Author), Henderson, J.M (Author), Hill, E. (Author), Houston, M.E (Author), Ko, N. (Author), McReynolds, T. (Author), Smith, C. (Author), Ujita, A. (Author), Yousif-Rosales, S. (Author)
• Format: Article
• Language: English
• Published: Blackwell Publishing Inc. 2021
• Subjects: 3' untranslated region; 5' untranslated region; Article; cap 1; cap analog; capped RNA; CleanCap; controlled study; enzyme mechanism; gene expression; human; Humans; in vitro study; In Vitro Techniques; in vitro transcription; isolation and purification; messenger RNA; Protein Biosynthesis; protein synthesis; RNA analysis; RNA Cap Analogs; RNA capping; RNA purification; RNA stability; RNA Stability; RNA structure; RNA synthesis; RNA transcription; RNA, Messenger; synthesis
• Online Access: View Fulltext in Publisher

 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 The Authors. Basic Protocol 1: IVT with CleanCap. Basic Protocol 2: mRNA purification and analysis. © 2021 The Authors.