An Elongation- and Ligation-Based qPCR Amplification Method for the Radiolabeling-Free Detection of Locus-Specific N⁶...

The epitranscriptomic mark N⁶ -methyladenosine (m⁶ A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m⁶ A detection methods have precluded routine identification of m⁶ A marks at the single-site level in mRNA transcripts. Herein, we report a single-base elongation- and ligation-based qPCR amplification method (termed "SELECT") that exploits the ability of m⁶ A to hinder 1) the single-base elongation activity of DNA polymerases and 2) the nick ligation efficiency of ligases; SELECT employs qPCR for quantitation. Following optimization and validation, SELECT was applied on three highly relevant proof-of-concept cases: determining 1) if a putative m⁶ A site is m⁶ A-modified in mRNAs and lncRNAs from biological samples, 2) the m⁶ A fraction at biological sites, and 3) if a particular m⁶ A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m⁶ A marks with unprecedented ease.