5-hydroxymethylcytosine (5-hmC) is a DNA modification that is – along with its analog 5-methylcytosine (5-mC) – an important epigenetic marker associated with critical cellular functions such as stem cell differentiation and brain development. A specific detection of 5-hmC however, is not trivial since traditional sodium bisulfite sequencing does not distinguish between 5-mC and 5-hmC sites.
Song et al. reported an approach for the selective detection of 5-hmC residues in genomic DNA based on UDP-6-azide-glucose (UDP-6-N3-Glc) (Fig. 1). T4 Phage β-glucosyltransferase selectively transfers an azide-modified glucose moiety to the hydroxyl group of 5-hmC allowing detection via Copper-free strain-promoted alkyne-azide click chemistry (SPAAC) by
Figure 1 Workflow of 5-hmC detection. Step 1: T4 Phage ß-glycosyltransferase (T4 ß-GT)-mediated glycosylation and simultaneous azide-functionalization of 5-hmC residues using UDP-6-azide-glucose (UDP-6-N3-Glc). Step 2: Copper-free labeling of Azide (N3) with DBCO-containing detection molecule (DBCO-containing (Desthio)Biotin → Click Chemistry or DBCO-containing Fluorescent Dyes → Click Chemistry) forming a stable triazole moiety. Step 3: Detection, affinity purification or sequencing of 5-hmC containing DNA (modified according to ).
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 Song et al. (2016) Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation. PNAS 113(16):4339.