Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry
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Charlotte Vranken, Jochem Deen, Lieve Dirix, Tim Stakenborg, Wim Dehaen, Volker Leen, Johan Hofkens, Robert K. Neely
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).
|Tidsskrift||Nucleic Acids Research|
|Status||Udgivet - 1 apr. 2014|