Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).

Jaar van publicatie:2014

BOF-keylabel:ja

IOF-keylabel:ja

BOF-publication weight:10

CSS-citation score:2

Auteurs:International

Authors from:Government, Higher Education