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N  6 -Methyladenine (m 6 A) is the most abundant internal modification on mammalian mRNA. Very recently, m 6 A has been reported as a potentially important 'epigenetic' mark in eukaryotes. Until now, site-specific detection of m 6 A is technically very challenging. Here, we first reveal that m 6 A significantly hinders DNA- and RNA-directed DNA synthesis. Systematic investigations of 5'-triphosphates of a variety of 5-substituted 2'-deoxyuridine analogs in primer extension have been performed. In the current study, a quantitative analysis of m 6 A in the RNA or DNA context has been achieved, using Bst DNA polymerase catalyzed primer extension. Molecular dynamics study predicted that m 6 A in template tends to enter into and be restrained in the MGR region of Bst DNA polymerase, reducing conformational flexibility of the DNA backbone. More importantly, a site-specific determination of m 6 A in human ribosomal RNA (rRNA) with high accuracy has been afforded. Through a cumulative analysis of methylation alterations, we first reveal that significantly cancer-related changes in human rRNA methylation were present in patients with hepatocellular carcinoma.

N 6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens

N ⁶-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens