Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N⁶-Methyladenine in Human Cells

DNA N⁶-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [¹⁵N₅]-2'-deoxyadenosine ([¹⁵N₅]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [¹⁵N₅]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine ¹⁵N, and further utilizes the purine salvage pathway to generate mainly both [¹⁵N₄]-dA and [¹⁵N₄]-2'-deoxyguanosine ([¹⁵N₄]-dG) in mammalian genomes. However, [¹⁵N₅]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [¹⁵N₄]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.