N6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens

Abstract

N ⁶-Methyladenine (m⁶A) is the most abundant internal modification on mammalian mRNA. Very recently, m⁶A has been reported as a potentially important ‘epigenetic’ mark in eukaryotes. Until now, site-specific detection of m⁶A is technically very challenging. Here, we first reveal that m⁶A 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 m⁶A in the RNA or DNA context has been achieved, using Bst DNA polymerase catalyzed primer extension. Molecular dynamics study predicted that m⁶A 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 m⁶A 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.