Methyltransferase-directed covalent coupling of fluorophores to DNA

被引：17

作者：

Lauer, Milena Helmer ^{[1
,2
]}

Vranken, Charlotte ^{[1
]}

Deen, Jochem ^{[1
]}

Frederickx, Wout ^{[1
]}

Vanderlinden, Willem ^{[1
]}

Wand, Nathaniel ^{[3
]}

Leen, Volker ^{[1
]}

Gehlen, Marcelo H. ^{[2
]}

Hofkens, Johan ^{[1
]}

Neely, Robert K. ^{[3
]}

机构：

[1] Katholieke Univ Leuven, Dept Chem, Celestijnenlaan, B-3001 Heverlee, Belgium

[2] Univ Sao Paulo, Inst Chem Sao Carlos, Sao Paulo, Brazil

[3] Univ Birmingham, Sch Chem, Birmingham B15 2TT, W Midlands, England

来源：

CHEMICAL SCIENCE | 2017年 / 8卷 / 05期

基金：

欧洲研究理事会; 巴西圣保罗研究基金会; 英国工程与自然科学研究理事会;

关键词：

DEPENDENT METHYLTRANSFERASES; SINGLE-MOLECULE; ANALOGS; CYCLOADDITION; RESOLUTION; CHEMISTRY; BINDING;

D O I：

10.1039/c6sc04229e

中图分类号：

O6 [化学];

学科分类号：

0703 ;

摘要：

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).

引用

页码：3804 / 3811

页数：8

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