Deoxyribozyme-based Method for Site-specific Absolute Quantification of N6-methyladenosine Modification Fraction

N6-methyladenosine (m6A) is the most prevalent modified base in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRNA). Although candidate sites for m6A modification are identified at the transcriptomic level, site-specific quantification methods for m6A modifications are still limited. Herein, we present a facile method implementing deoxyribozyme that preferentially cleaves the unmodified RNA. We leverage reverse transcription and real-time quantitative PCR along with key control experiments to quantify the absolute methylation fraction of specific m6A sites. We validate the accuracy of the method using synthetic RNA with controlled methylation fraction and apply our method on 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, expanding the current toolkit for studying RNA modifications.