An Elongation‐ and Ligation‐Based qPCR Amplification Method for the Radiolabeling‐Free Detection of Locus‐Specific N6‐Methyladenosine Modification
Abstract
The epitranscriptomic mark N 6 -methyladenosine (m 6 A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m 6 A detection methods have precluded routine identification of m 6 A marks at the single-site level in mRNA transcripts. Herein, we report a single-base elongation- and ligation-based qPCR amplification method (termed "SELECT") that exploits the ability of m 6 A to hinder 1) the single-base elongation activity of DNA polymerases and 2) the nick ligation efficiency of ligases; SELECT employs qPCR for quantitation. Following optimization and validation, SELECT was applied on three highly relevant proof-of-concept cases: determining 1) if a putative m 6 A site is m 6 A-modified in mRNAs and lncRNAs from biological samples, 2) the m 6 A fraction at biological sites, and 3) if a particular m 6 A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m 6 A marks with unprecedented ease.
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The detection of m6A at targeted sites was based on the SELECT method previously developed by Xiao et al. (2018).
“The detection of m6A at targeted sites was based on SELECT method previously developed by Xiao et al., (2018).”
From METTL3 uncouples chromatin accessibility from transcription during retinal development (2025) · cites this paper as Xiao et al., (2018)
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N6-methyladenosine-dependent regulation of messenger RNA stability
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Self-citationComprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons
Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., & Jaffrey, S. R. (2012) · Cell · doi:10.1016/j.cell.2012.05.003 · Cited by 4,223 · see citing works
N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO
Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y.-G., et al. (2011) · Nature Chemical Biology · doi:10.1038/nchembio.687 · Cited by 4,106 · see citing works
Self-citationALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility
Zheng, G., Dahl, J. A., Niu, Y., Fedorcsak, P., Huang, C.-M., Li, C. J., Vågbø, C. B., Shi, Y., Wang, W.-L., Song, S.-H., et al. (2013) · Molecular Cell · doi:10.1016/j.molcel.2012.10.015 · Cited by 3,550 · see citing works
Self-citationRecognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation
Huang, H., Weng, H., Sun, W., Qin, X., Shi, H., Wu, H., Zhao, B. S., Mesquita, A., Liu, C., Yuan, C. L., et al. (2018) · Nature Cell Biology · doi:10.1038/s41556-018-0045-z · Cited by 2,997 · see citing works
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Absolute quantification of single-base m6A methylation in the mammalian transcriptome using GLORI
Liu, C., Sun, H., Yi, Y., Shen, W., Li, K., Xiao, Y., Li, F., Li, Y., Hou, Y., Lu, B., et al. (2023) · Nature Biotechnology · doi:10.1038/s41587-022-01487-9 · Cited by 386 · see citing works
Self-citationAVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease
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Self-citationMETTL3 uncouples chromatin accessibility from transcription during retinal development
Xu, J., Huang, Y., Han, Z., Li, Q., Liu, J., & Rao, R. C. (2025) · Stem Cell Reports · doi:10.1016/j.stemcr.2025.102690 · Cited by 1 · see citing works
- Authors
- Xiao, Y., Wang, Y., Tang, Q., Wei, L., Zhang, X., & Jia, G.
- Affiliations
- Peking University (CN)
- Department of Chemical Biology Synthetic and Functional Biomolecules Center Beijing National Laboratory for Molecular Sciences Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education College of Chemistry and Molecular Engineering Beijing Advanced Innovation Center for Genomics Peking University Beijing 100871 China
- Corresponding author
- Jia, Guifang
- Venue
- Angewandte Chemie International Edition
- Publisher
- Wiley
- Published
- 2018-11-08
- Volume
- 57
- Issue
- 49
- Pages
- 15995-16000
- ISSN
- 1433-7851 (ISSN-L)
- 1521-3773
- OpenAlex
- W2897520171
- Cited by
- 340
- References
- 5
- Citations by year
- 2026: 41 · 2025: 60 · 2024: 79 · 2023: 49 · 2022: 45 · 2021: 32 · 2020: 21 · 2019: 12 · 2018: 1
- FWCI
- 7.91
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Angewandte Chemie International Edition (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Ministry of Science and Technology of the People's Republic of China (MOST2016YFC0900302, 2017YFA0505201)
- National Natural Science Foundation of China (21432002, 21822702)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- N6-methyladenosine, epitranscriptomics, long non-coding RNA
- MeSH terms
- Adenosine, DNA-Directed DNA Polymerase, RNA, Messenger, Real-Time Polymerase Chain Reaction
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