Cap 1 Messenger RNA Synthesis with Co‐transcriptional CleanCap ® Analog by In Vitro Transcription
Open accessAbstract
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. This article was corrected on 22 November 2021 and 26 July 2022 . See the end of the full text for details. Basic Protocol 1 : IVT with CleanCap Basic Protocol 2 : mRNA purification and analysis
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The CleanCap® cap analogs can be co-transcribed with the target mRNA to obtain the cap1 structure, thus solving the issues of low efficiency and high enzyme costs of traditional capping methods.
“The current new generation of cap analogs is the CleanCap® cap analogs developed by TriLink BioTechnologies (San Diego, CA, USA),126 which can be co-transcribed with the target mRNA to obtain the cap1 structure, thus solving the issues of low efficiency and high enzyme costs of traditional capping methods.”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [126]
The use of CleanCap® cap analogs reduces the probability of reverse capped, uncapped, and cap0 intermediates, and the co-transcribed mRNA only possesses a cap1 structure, therefore capping rate can be 90% or higher.
“The use of CleanCap® cap analogs reduces the probability of reverse capped, uncapped, and cap0 intermediates, and the co-transcribed mRNA only possesses a cap1 structure, therefore capping rate can be 90% or higher.52,126”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [126]
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- Authors
- Henderson, J. M., Ujita, A., Hill, E., Yousif‐Rosales, S., Smith, C., Ko, N., McReynolds, T., Cabral, C. R., Escamilla‐Powers, J. R., & Houston, M. E.
- Affiliations
- TriLink BioTechnologies (United States) (US)
- Corresponding author
- Henderson, Jordana M.
- Venue
- Current Protocols
- Publisher
- Wiley
- Published
- 2021-02
- Volume
- 1
- Issue
- 2
- Pages
- e39-e39
- ISSN
- 2691-1299 (ISSN-L)
- OpenAlex
- W3127832258
- Cited by
- 231
- References
- 38
- Citations by year
- 2026: 39 · 2025: 53 · 2024: 49 · 2023: 45 · 2022: 36 · 2021: 9
- FWCI
- 10.90
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Current Protocols (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- mRNA therapeutics, in vitro transcription, mRNA synthesis, mRNA purification, mRNA stability, mRNA vaccines, SARS-CoV-2
- MeSH terms
- Humans, RNA Cap Analogs, RNA, Messenger, Protein Biosynthesis, RNA Stability, In Vitro Techniques
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