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Quantification and Single-Base Resolution Analysis of N1-Methyladenosine in mRNA by Ligation-Assisted Differentiation.
Anal Chem ; 92(3): 2612-2619, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31948230
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.





Texto completo: Disponível Coleções: Bases de dados internacionais Base de dados: MEDLINE Idioma: Inglês Revista: Anal Chem Ano de publicação: 2020 Tipo de documento: Artigo