MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N < sup > 6 < /sup > -methyladenosine RNA

Anal Chim Acta. 2024 May 15;1303:342532. doi: 10.1016/j.aca.2024.342532. Epub 2024 Mar 25.ABSTRACTN6-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.PMID:38609270 | DOI:10.1016/j.aca.2024.342532

Source: Analytica Chimica Acta - April 12, 2024 Category: Chemistry Authors: Guobin Han Qiuyuan Lin Jia Yi Qian Lyu Qingwei Ma Liang Qiao Source Type: research