School of Chemistry, Tel Aviv University, Tel Aviv
Hydroxymethylation of genomic cytosine (5hmC) is a chemical modification induced through the oxidation of methyl cytosine by the Tet family of enzymes. Modified bases have been detected at various levels across different tissues and cell types indicating a regulatory mechanism for 5hmC maintenance. With extreme recent attention from the epigenetic, stem cell and cancer communities, it is recognized as an important epigenetic marker linked to development and disease. Characterizing the distribution of hydroxymethylated bases relies on the development of methods for quantification and mapping of 5hmC residues. We take advantage of the sensitivity inherent to optical detection in order to develop a method for investigating 5hmC DNA content down to the single molecule level. To facilitate optical detection, we utilize a viral glucosyltranferase to transfer an azido modified UDP-glucose onto the hydroxymethylated cytosine. A click reaction is used to covalently attach a fluorescent reporter molecule to azido-glucosyl-5hmC. We show that sensitive quantification of hydroxymethylated DNA is achieved by a simple light absorption measurement and demonstrate the potential of single molecule optical mapping to reveal hydroxymethylation patterns on long genomic DNA fragments. Single molecule imaging of extended lambda phage genomes that are hydroxymethylated at specific sites reveals individual fluorescent labels at expected positions along the genome
