Chemo-Enzymatic Labelling of the Epigenetic DNA Modification 5-Hydroxymethylcytosine

The field of Epigenetics focuses on DNA and chromatin modifications not encoded in the DNA sequence. 5-Methylcytosine (5-mC), DNA methylation, is known to play a key role in diseases and differentiation mechanisms. It recently has been shown that 5-mC is oxidized to 5-hydroxymethylcytosine (5-hmC) in an endogenous enzymatic reaction. 5-hmC, displays tissue specific distribution and has been related to gene expression. For labeling purpose, 5-hmC can be selectively glycosylated by the β-glucosyltransferase enzyme (β-GT), originated from the T4 bacteriophage; using a synthetic substrate that enables fluorescent tagging of 5-hmC residues via click chemistry. This approach has not been broadly adopted due to the challenging synthesis and limited commercial availability of the glycosylation substrate 6-N3-UDPG. This work focused on finding a solution for this problem.