Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS
Abstract
A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.
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Determining mRNA capping efficiency is critical for quality control of mRNA vaccines, which is usually determined by liquid chromatography and mass spectrometry (LC-MS).
“As a result, determining mRNA capping efficiency is critical for quality control of mRNA vaccines, which is usually determined by liquid chromatography and mass spectrometry (LC-MS).467”
From Advances in COVID-19 mRNA vaccine development (2022) · cites this paper as [467]
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Advances in COVID-19 mRNA vaccine development
Fang, E., Liu, X., Li, M., Zhang, Z., Song, L., Zhu, B., Wu, X., Liu, J., Zhao, D., & Li, Y. (2022) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-022-00950-y · Cited by 601 · see citing works
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Advancing mRNA technologies for therapies and vaccines: An African context
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- Authors
- Beverly, M., Dell, A., Parmar, P., & Houghton, L.
- Corresponding author
- Beverly, Michael
- Venue
- Analytical and Bioanalytical Chemistry
- Published
- 2016-05-18
- Volume
- 408
- Issue
- 18
- Pages
- 5021-5030
- ISSN
- 1618-2642 (ISSN-L)
- 1618-2650
- OpenAlex
- W2395331380
- Cited by
- 83
- References
- 31
- Citations by year
- 2026: 14 · 2025: 15 · 2024: 11 · 2023: 14 · 2022: 18 · 2021: 2 · 2020: 3 · 2019: 3 · 2018: 1 · 2017: 2
- FWCI
- 0.99
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Analytical and Bioanalytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Defense Advanced Research Projects Agency (HR-0011-13-3-0003.)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
- Keywords
- RNase H cleavage, label-free detection, streptavidin-coated magnetic beads, pseudouridine, 5-methylcytidine
- MeSH terms
- Chromatography, Liquid, RNA Caps, RNA, Messenger, Sensitivity and Specificity, Mass Spectrometry, Staining and Labeling, Reproducibility of Results, Molecular Probes, Molecular Probe Techniques, Ribonuclease H, Sequence Analysis, RNA
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