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

Hospital-based RNA Therapeutics

“…To ensure RNA translation, 7-methylguanylate cap must be incorporated into mRNA either posttranscriptionally with vaccinia capping enzyme (Martin and Moss 1975) or co-transcriptionally through the addition of an anti-reverse cap analog (ARCA) or CleanCap reagent (Henderson et al 2021). Different modified NTPs, such as pseudouridine or 5-methyluridine, may be incorporated into mRNA construct by adding them to IVT reaction mixture.…”

“…Purification methods have reduced the immunogenicity of IVT mmRNAs and have enabled the development of mRNA therapeutics that will be applicable to many diseases. Precipitation (Henderson et al 2021) and liquid chromatography-based methods (Karikó et al 2011) can be employed to purify RNA from enzymes, free NTPs, residual DNA, truncated RNA fragments, and double-stranded RNA. At small scales, preparative polyacrylamide gel electrophoresis is a powerful and commonly used tool to separate the desired RNA products from abortive products or other impurities (Green and Sambrook 2021).…”

Vaccines against COVID-19: Priority to mRNA-Based Formulations

“…Improvements of the ivt mRNA production, design, and structure these last 20 y have transformed this molecule into a very efficient genetic vehicle for vaccines and therapies [36,37]. Of note, a revolutionary Cap analog named CleanCap has largely improved the production and efficacy of synthetic mRNA [38]. The ivt mRNA encodes a single antigen, limiting the risk of triggering immunity against irrelevant antigens, as can be seen with proteins (i.e., contaminants, misfolded proteins) or adenoviruses (vector proteins).…”

An Update on mRNA-Based Viral Vaccines

“…The 5′ capping in mature mRNA is required for protection of mRNA from degradation, facilitating recruitment of the ribosomes, gene expression, and self- versus non-self-identification. Several variations of 5′ cap structures have been found to exist in nature [ 36 , 37 ]. The 5′ cap of the eukaryotic mRNA contains 7-methylguanosine (m7G) through a 5′-5′-triphosphate bridge (m7GpppN) via a series of enzymatic capping reactions involving RNA triphosphatase, guanosyltransferase, and S-adenosyl methionine [ 38 ].…”

“…To avoid this drawback, enhanced translation efficacy was reported with an anti-reverse cap analog (ARCA: 3′-O-Me-m7GpppG) where the 3′-OH hydrophilic group was methylated to block reverse incorporation by RNA polymerase [ 40 ]. A new strategy of co-transcription was to specifically add a natural 5′ cap1 structure to the start site during IVT reactions using a CleanCap kit (TriLink Biotechnology), simplifying the 5′ capped mRNA production by T7 polymerase in vitro reactions [ 37 , 41 ], which has become a commonly used capping method resulting in high translation and low reactogenicity. An approach of CleanCap was used for 5′ capping of COVID-19 BNT162b1 mRNA vaccines [ 42 ].…”