Direct quantification of N⁶-methyladenosine fractions at specific site in RNA based on deoxyribozyme mediated...

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N⁶-methyladenosine (m⁶A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m⁶A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m⁶A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m⁶A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m⁶A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m⁶A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m⁶A fractions in real biological samples, even in low abundance RNA biomarkers.