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

ABSTRACT N 6 -methyladenosine (m 6 A) is the most prevalent modified base in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRNA). Although candidate sites for m 6 A modification are identified at the transcriptomic level, site-specific quantification methods for m 6 A 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 m 6 A 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 m 6 A fraction at specific loci, expanding the current toolkit for studying RNA modifications.