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

Charlotte Vranken; Jochem Deen; Lieve Dirix; Tim Stakenborg; Wim Dehaen; Volker Leen; Johan Hofkens; Robert K. Neely

doi:10.1093/nar/gkt1406

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

Charlotte Vranken, Jochem Deen, Lieve Dirix, Tim Stakenborg, Wim Dehaen, Volker Leen, Johan Hofkens, Robert K. Neely

Kemisk Institut

36 Citationer (Scopus)

Abstract

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).

Originalsprog	Engelsk
Artikelnummer	e50
Tidsskrift	Nucleic Acids Research
Vol/bind	42
Udgave nummer	7
ISSN	0305-1048
DOI	https://doi.org/10.1093/nar/gkt1406
Status	Udgivet - 1 apr. 2014

Adgang til dokumentet

10.1093/nar/gkt1406

Citationsformater

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title = "Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry",

abstract = "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). ",

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T1 - Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

AU - Vranken, Charlotte

AU - Deen, Jochem

AU - Dirix, Lieve

AU - Stakenborg, Tim

AU - Dehaen, Wim

AU - Leen, Volker

AU - Hofkens, Johan

AU - Neely, Robert K.

PY - 2014/4/1

Y1 - 2014/4/1

N2 - 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).

AB - 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).

U2 - 10.1093/nar/gkt1406

DO - 10.1093/nar/gkt1406

M3 - Journal article

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