HPLC purification of in vitro transcribed long RNA
Abstract
In vitro transcription of DNA with phage RNA polymerases is currently the most efficient method to produce long sequence-specific RNA. While the reaction can yield large quantities of RNA, it contains impurities due to various unwanted activities of the polymerases. Here, we described an easily performed HPLC purification that removes multiple contaminants from in vitro transcribed RNA and is scalable. The purified RNA is translated at much greater levels, especially in primary cells and in vivo. HPLC purification of RNA containing modified nucleosides that suppress RNA-mediated activation of innate immune sensors leads to a non-immunogenic RNA with superior translational capacity.
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HPLC, anion exchange and affinity chromatography are used in the purification process to remove truncated transcripts.
“HPLC, anion exchange and affinity chromatography, are used in the purification process to remove truncated transcripts (Weissman et al. 2013).”
From Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency (2022) · cites this paper as Weissman et al. 2013
Purification of IVT mRNA using cellulose, HPLC, FPLC, oligo(dT) purification, or TFF can further improve mRNA quality.
“Further improvement of mRNA quality can be made with purification of IVT mRNA using cellulose,39 high-pressure liquid chromatography (HPLC),40 fast protein liquid chromatography (FPLC),41 oligo(dT) purification,3 or tangential flow filtration (TFF).42,43”
From COVID-19 mRNA vaccines: Platforms and current developments (2022) · cites this paper as [40]
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- Authors
- Weissman, D., Pardi, N., Muramatsu, H., & Karikó, K.
- Affiliations
- University of Pennsylvania (US)
- Corresponding author
- Weissman, Drew
- Venue
- Methods in molecular biology
- Publisher
- Humana Press
- Published
- 2012-11-08
- Volume
- 969
- Pages
- 43-54
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W2417233462
- Cited by
- 180
- References
- 32
- Citations by year
- 2026: 10 · 2025: 22 · 2024: 24 · 2023: 27 · 2022: 21 · 2021: 29 · 2020: 17 · 2019: 11 · 2018: 10 · 2017: 3 · 2016: 3 · 2015: 1 · 2014: 2
- FWCI
- 1.64
- Type
- book chapter
- Language
- en
- Open access
- closed
- Licence
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Heart, Lung, and Blood Institute (R42HL087688, HL87688)
- National Institute of Allergy and Infectious Diseases (AI090788, R01AI050484, AI050484, R01AI090788)
- National Institute of Dental and Craniofacial Research (DE019059, R21DE019059)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- in vitro transcription, RNA purification, HPLC, phage RNA polymerase, RNA translation, modified nucleosides, innate immune activation, immunogenicity, primary cells
- MeSH terms
- Chromatography, High Pressure Liquid, DNA, Nucleosides, RNA, DNA-Directed RNA Polymerases, Transcription, Genetic
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