Deoxyribozyme-based method for absolute quantification of N6-methyladenosine fractions at specific sites of RNA

N-6-Methyladenosine (m(6)A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m(6)A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m(6)A modification levels are still limited. Herein, we present a facile method implementing a deoxyribozyme, VMC10, which preferentially cleaves the unmodified RNA. We leveraged reverse transcription and real-time quantitative PCR along with key control experiments to quantify the methylation fraction of specific m(6)A sites. We validated the accuracy of this method with synthetic RNA in which methylation fractions ranged from 0 to 100% and applied our method to several endogenous sites that were previously identified in sequencing-based studies. This method provides a time- and cost-effective approach for absolute quantification of the m(6)A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.