Issue 5, 2017

Methyltransferase-directed covalent coupling of fluorophores to DNA

Abstract

We report an assay for determining the number of fluorophores conjugated to single plasmid DNA molecules and apply this to compare the efficiency of fluorophore coupling strategies for covalent DNA labelling. We compare a copper-catalyzed azide–alkyne cycloaddition reaction, amine to N-hydroxysuccinimidyl ester coupling reaction and strain-promoted azide–alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N-hydroxysuccinimidyl ester coupling reaction to the copper-catalyzed azide–alkyne cycloaddition and found the highest degree of DNA labelling with the strain-promoted azide–alkyne cycloaddition reaction. We also examined the effect of labelling on the DNA structure using atomic force microscopy. We observe no distortions or damage to the DNA that was labeled using the amine to N-hydroxysuccinimidyl ester and strain-promoted azide–alkyne cycloaddition coupling reactions. This was in contrast to the copper-catalyzed azide–alkyne cycloaddition reaction, which, despite the use of copper-coordinating ligands in the labelling mixture, leads to some structural DNA damage (single-stranded DNA breaks).

Graphical abstract: Methyltransferase-directed covalent coupling of fluorophores to DNA

Supplementary files

Article information

Article type
Edge Article
Submitted
21 सितम्बर 2016
Accepted
13 मार्च 2017
First published
14 मार्च 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2017,8, 3804-3811

Methyltransferase-directed covalent coupling of fluorophores to DNA

M. H. Lauer, C. Vranken, J. Deen, W. Frederickx, W. Vanderlinden, N. Wand, V. Leen, M. H. Gehlen, J. Hofkens and R. K. Neely, Chem. Sci., 2017, 8, 3804 DOI: 10.1039/C6SC04229E

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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