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

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