Methyl roadblock rolling circle amplification strategy enables an early screening of cervical cancer by detecting locus-specific N6-methyladenosine in mRNA

Early diagnosis is an important strategy for cancer prevention, and tumor marker determination has become a useful option. N-6-methyladenosine (m(6)A) is associated with the occurrence of various malignant tumors, thus being considered a crucial biomarker. However, precise analytical technology for m(6)A detection in locus-specific mRNA remains a challenging task. In this study, we developed a methyl roadblock rolling circle amplification reaction (MRRCA) strategy for m(6)A detection. The m(6)A can affect the stability of m(6)A-T base pairs and delay the extension of padlock probe, thus resulting in less efficiency in creating circular DNA structures, which produces different amounts of ssDNA long-stranded products. The fluorescence signal discrepancy between m(6)A and A can be detected after staining, enabling the discrimination of m(6)A. We demonstrate that low abundance m(6)A in RNAs can be accurately quantified with 100 ng of sample input via laboratory thermostatic equipment. Besides this, the m(6)A content of CENPK mRNA in different cells and clinical blood samples were quantified. It was found that the m(6)A level in HeLa cells was six times greater than that in normal cells. Similarly, compared with normal blood sample, the m(6)A content was similar to 1.8-fold greater in precancerous cervical sample and similar to 2.3-fold greater in confirmed cervical cancer sample. The investigation confirms a high expression of m(6)A in cervical cancer, and indicates its significant application in the early detection of cervical cancer in clinic. Moreover, the MRRCA strategy can be used to screen methyltransferase inhibitors and provides new opportunities for the development of antitumor drugs.