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

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.