Deoxyribozyme-based method for absolute quantification of N 6-methyladenosine fractions at specific sites of RNA.

N 6-Methyladenosine (m6A) is the most prevalent modified base in eukaryotic mRNA and long noncoding RNA. Although candidate sites for the m6A modification are identified at the transcriptomic level, methods for site-specific quantification of absolute m6A 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 m6A 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 m6A fraction at specific loci, with the potential for multiplexed quantifications, expanding the current toolkit for studying RNA modifications.