AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in...

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N⁶,N⁶-Dimethyladenosine (m^6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m^6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m^6,6A) method for the site-specific detection and quantification of m^6,6A in RNA. The N⁶,N⁶-dimethyl groups in m^6,6A can cause reverse transcription to stall at the m^6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m^6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m^6,6A in RNA. Using the AD-m^6,6A method, we successfully detected and quantified m^6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m^6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m^6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m^6,6A in RNA, which can aid in uncovering the functions of m^6,6A in human diseases.