title: One-Pot Chemoenzymatic Cascade for Labeling of the Epigenetic Marker 5-Hydroxymethylcytosine.
abstract: The epigenetic DNA modification 5-hydroxymethylcytosine (5-hmC) is important for the regulation of gene expression during development and in tumorigenesis. 5-hmC can be selectively glycosylated by T4 β-glucosyltransferase (β-GT); introduction of an azide on the attached sugar provides a chemical handle for isolation or fluorescent tagging of 5-hmC residues by click chemistry. This approach has not been broadly adopted because of the challenging synthesis and limited commercial availability of the glycosylation substrate, 6-deoxy-6-azido-α-D-glucopyranoside. We report the enzyme-assisted synthesis of this precursor by the uridylyltransferase from Pasteurella multocida (PmGlmU). We were able to directly label 5-hmC in genomic DNA by an enzymatic cascade involving successive action of PmGlmU and β-GT. This is a facile and cost-effective one-pot chemoenzymatic methodology for 5-hmC analysis.
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Gil
authors.initials: G
authors.lastname: Nifker
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Michal
authors.initials: M
authors.lastname: Levy-Sakin
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Yifat
authors.initials: Y
authors.lastname: Berkov-Zrihen
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Tamar
authors.initials: T
authors.lastname: Shahal
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Tslil
authors.initials: T
authors.lastname: Gabrieli
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Micha
authors.initials: M
authors.lastname: Fridman
authors.affiliation: Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel Aviv 6997801 (Israel).
authors.forename: Yuval
authors.initials: Y
authors.lastname: Ebenstein
keywords: DNA methylation; chemoenzymatic synthesis; fluorescent probes; hydroxymethylcytosine; single molecules; sugar nucleotides