Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N(6)-methyladenosine

N6-methyladenosine (m(6)A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m(6)A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m(6)A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m(6)A-Rol-LAMP, to verify putative m(6)A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m(6)A sites on targets, they are converted to circular form by DNA ligase in the absence of m(6)A modification, while m(6)A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m(6)A. Following optimization and validation, m(6)A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m(6)A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m(6)A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m(6)A, which can simply, quickly, sensitively, specifically, and visually determine putative m(6)A modification on RNA.