MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N⁶-methyladenosine RNA

N⁶-methyladenosine (m⁶A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m⁶A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m⁶A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m⁶A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m⁶A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64-106.93%. The present assay provides a novel platform for the analysis of site-specific m⁶A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.