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).
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium, Life Science Technologies, Imec, Kapeldreef 75, 3001 Heverlee, Belgium and Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Charlotte
C
Vranken
Jochem
J
Deen
Lieve
L
Dirix
Tim
T
Stakenborg
Wim
W
Dehaen
Volker
V
Leen
Johan
J
Hofkens
Robert K
RK
Neely
D000478:Alkylation; D057930:Click Chemistry; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D015254:DNA Modification Methylases / Q000378:metabolism*; D005456:Fluorescent Dyes; D023281:Genomics / Q000379:methods*; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry
D005456:Fluorescent Dyes; D012436:S-Adenosylmethionine; D004247:DNA; D015254:DNA Modification Methylases