Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine

N6-methyladenosine (m⁶A) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific m⁶A marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific m⁶A detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed “m⁶A-MazF-SAT”. Mechanically, MazF fails to cleave the A (m⁶A) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of m⁶A existence. After the condition optimization, m⁶A-MazF-SAT can significantly, accurately, and rapidly detect the m⁶A-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% m⁶A at the fmol level. In addition, m⁶A-MazF-SAT can quantify the abundance of target m⁶A in biological samples and can be used for the inhibitor selection of m⁶A-related enzymes. Together, we offer a new approach to detect locus-specific m⁶A both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.