Methyltransferase-directed covalent coupling of fluorophores to DNA

Milena Helmer Lauer, Charlotte Vranken, Jochem Deen, Nathaniel Wand, Wout Frederickx, Willem Vanderlinden, Volker Leen, Marcelo Gehlen, Johan Hofkens, Robert Neely

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)
165 Downloads (Pure)

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 chemistries for covalent DNA labelling. We compare the copper-catalyzed azide-alkyne cycloaddition reaction, the amine to N-hydroxysuccinimidyl ester coupling reaction and the strain-promoted azide-alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N-hydroxysuccinimidyl ester chemistry to 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 of 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).
Original languageEnglish
Pages (from-to)3804-3811
Number of pages8
JournalChemical Science
Volume8
Issue number5
Early online date14 Mar 2017
DOIs
Publication statusPublished - 1 May 2017

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