Quantification and Single-Base Resolution Analysis of N1-Methyladenosine in mRNA by Ligation-Assisted Differentiation

RNA modification, such as N1-methyladenosine (m¹A), affects the secondary structure of RNA and its ability to recognize specific reader proteins. Methods for detecting site-specific m¹A are in demand. We report here a ligation-assisted differentiation approach for quantitative detection of m¹A in mRNA with single-base resolution. The methyl group in m¹A disrupts the Watson-Crick base pairing with uridine, resulting in a lower ligation efficiency of certain ligases and lower amounts of ligation products. Detection of the ligation products using quantitative real-time PCR provided site-specific evaluation of m¹A. We first screened appropriate ligase and found that T3 DNA ligase offered the best discrimination between m¹A and adenosine. We successfully detected and quantified m¹A at position 1674 of bromodomain containing 2 (BRD2) mRNA from HEK293T cells. In lung carcinoma tissues, the level of m¹A at position 1674 of BRD2 mRNA was significantly decreased compared to the tumor-adjacent normal tissues, suggesting that site-specific m¹A may be involved in carcinogenesis.