Quantification and single-base resolution analysis of N1-methyladenosine in mRNA by ligation-assisted differentiation.

RNA modification, such as N1-methyladenosine (m(1)A), affects the secondary structure of RNA and its ability to recognize specific reader proteins. Methods for detecting site-specific m(1)A are in demand. We report here a ligation-assisted differentiation approach for quantitative detection of m(1)A in mRNA with singlebase resolution. The methyl group in m(1)A 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 m(1)A. We first screened appropriate ligase and found that T3 DNA ligase offered the best discrimination between m(1)A and adenosine. We successfully detected and quantified m(1)A at position 1674 of bromodomain containing 2 (BRD2) mRNA from HEK293T cells. In lung carcinoma tissues, the level of m(1)A at position 1674 of BRD2 mRNA was significantly decreased compared to the tumor-adjacent normal tissues, suggesting that site-specific m(1)A may be involved in carcinogenesis.