One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N⁶‑Methyladenosine Modifications

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.