Quantitative LC–MS Provides No Evidence for m⁶dA or m⁴dC in the Genome of Mouse Embryonic Stem Cells and Tissues

Abstract

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m⁵ dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N⁶ -methyldeoxyadenosine (m⁶ dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N⁴ -methyldeoxycytidine (m⁴ dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m⁵ dC, hmdC, fdC, and cadC, but we were unable to detect either m⁴ dC or m⁶ dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.