An Elongation‐ and Ligation‐Based qPCR Amplification Method for the Radiolabeling‐Free Detection of Locus‐Specific <i>N</i>⁶‐Methyladenosine Modification

Abstract

Abstract The epitranscriptomic mark N 6 ‐methyladenosine (m 6 A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m 6 A detection methods have precluded routine identification of m 6 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 m 6 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 m 6 A site is m 6 A‐modified in mRNAs and lncRNAs from biological samples, 2) the m 6 A fraction at biological sites, and 3) if a particular m 6 A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m 6 A marks with unprecedented ease.