Quantification and Single-Base Resolution Analysis of N 1-Methyladenosine in mRNA by Ligation-Assisted Differentiation.

RNA modification, such as N 1-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 single-base 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.