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

N⁶-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.