An Elongation‐ and Ligation‐Based qPCR Amplification Method for the Radiolabeling‐Free Detection of Locus‐Specific N⁶‐Methyladenosine Modification

Abstract

The epitranscriptomic mark N6‐methyladenosine (m6A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m6A detection methods have precluded routine identification of m6A 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 m6A 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 m6A site is m6A‐modified in mRNAs and lncRNAs from biological samples, 2) the m6A fraction at biological sites, and 3) if a particular m6A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m6A marks with unprecedented ease.

Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates

“…As an independent validation, we applied a non-antibody-based methodology to quantify the site-specific m 6 A editing. The recently reported SELECT method exploits the ability of m 6 A to hinder the single-base elongation activity of DNA polymerases and the nick ligation efficiency of ligases 28 ( Supplementary Fig. 3a ).…”

The 18S rRNA m 6 A methyltransferase METTL 5 promotes mouse embryonic stem cell differentiation

“…For quantitatively detecting the m 6 A status in 18S rRNA A 1,832 and A 1,825 (as input control) locus, the SELECT (single‐base elongation‐ and ligation‐based PCR amplification method) was performed as described (Xiao et al , ). In brief, we used 1 ng total RNA extracted from different cells, 40 nM up/down primer and 5 μM dNTP, 1 × CutSmart buffer in the 17 μl reaction mixture, which were annealed at a temperature gradient.…”

Single-base mapping of m 6 A by an antibody-independent method

“…The recently developed high-sensitivity liquid chromatography-tandem mass spectrometry and blotting methods relying on antibodies were widely used to quantify the overall m 6 A level. To detect individual sites, methyl-sensitive ligase has been applied to confirm the methylation status of specific adenosines (16,17), while the method called SCARLET (site-specific cleavage and radioactive-labeling followed by ligationassisted extraction and thin-layer chromatography) can quantify the methylation level of individual m 6 A site (11). Other methods were developed to identify m 6 A in single-base resolution during reverse transcription, taking advantage of m 6 A-sensitive reverse transcriptase (18,19), chemoenzymatic substitution of the N 6 -methyl group (20), or selective dTTP (deoxythymidine triphosphate) analog such as 4SedTTP (21).…”