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Updated: Aug 17, 2026
Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Published on: November 22, 2014
One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications
Xiaochen Ma1, Yuqing Xia1, Shizheng Wang1
1Department of Chemistry, Faculty of Environment and Life Science, Beijing University of Technology, Beijing 100124, China.
Abstract:
N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.
Insights
Researchers developed a new method to precisely detect N⁶-Methyladenosine (m⁶A) RNA modifications. This ultrasensitive assay offers single-base resolution for accurate m⁶A quantification in cells, aiding disease diagnosis.
Area of Science:
- Molecular Biology
- Epigenetics
- Biochemistry
Background:
- N⁶-Methyladenosine (m⁶A) is a key RNA modification regulating mRNA metabolism in mammals.
- Existing methods lack precision in quantifying m⁶A at specific sites.
Purpose of the Study:
- To develop a robust method for ultrasensitive, site-specific quantification of m⁶A RNA.
- To enable single-base resolution detection of m⁶A modifications.
Main Methods:
- Screening of a Bst 2.0 warm start DNA polymerase for m⁶A discrimination.
- Development of an isothermal and ultrasensitive m⁶A RNA detection assay.
- Application of the assay to single-cell analysis.
Main Results:
- The assay achieves a detection limit of approximately 0.02 amol.
- Quantitative accuracy was validated in real cell samples.
- Single-cell analysis revealed significant differences in MALAT1 m⁶A levels between glioblastoma and oligodendrocyte cells.
Conclusions:
- The developed method provides a highly sensitive tool for site-specific m⁶A detection and quantification.
- This technology is expected to advance precise disease diagnosis and the study of epigenetic transcriptional regulation.
