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. 2021 Feb;1(2):e39.
doi: 10.1002/cpz1.39.

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

Jordana M Henderson  1 , Andrew Ujita  1 , Elizabeth Hill, Sally Yousif-Rosales  1 , Cory Smith  1 , Nicholas Ko  1 , Taylor McReynolds  1 , Charles R Cabral  1 , Julienne R Escamilla-Powers  1 , Michael E Houston  1
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Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription

Jordana M Henderson et al. Curr Protoc. 2021 Feb.

Erratum in

Abstract

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.

Keywords: CleanCap; cap 1; cap analog; in vitro transcription; messenger RNA.

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