Lauer Milena Helmer, Vranken Charlotte, Deen Jochem, Frederickx Wout, Vanderlinden Willem, Wand Nathaniel, Leen Volker, Gehlen Marcelo H, Hofkens Johan, Neely Robert K
Department of Chemistry , KU Leuven , Celestijnenlaan , 3001 Heverlee , Belgium.
Institute of Chemistry of São Carlos , University of São Paulo , Brazil.
Chem Sci. 2017 May 1;8(5):3804-3811. doi: 10.1039/c6sc04229e. Epub 2017 Mar 14.
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 -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 -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 -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).
我们报告了一种用于确定与单个质粒DNA分子缀合的荧光团数量的测定方法，并将其应用于比较共价DNA标记的荧光团偶联策略的效率。我们比较了用于荧光DNA标记的铜催化的叠氮化物-炔烃环加成反应、胺与N-羟基琥珀酰亚胺酯偶联反应以及应变促进的叠氮化物-炔烃环加成反应。我们发现从胺与N-羟基琥珀酰亚胺酯偶联反应到铜催化的叠氮化物-炔烃环加成反应，标记效率有所提高，并且应变促进的叠氮化物-炔烃环加成反应实现了最高程度的DNA标记。我们还使用原子力显微镜检查了标记对DNA结构的影响。我们观察到，使用胺与N-羟基琥珀酰亚胺酯以及应变促进的叠氮化物-炔烃环加成偶联反应标记的DNA没有变形或损伤。这与铜催化的叠氮化物-炔烃环加成反应形成对比，尽管在标记混合物中使用了铜配位配体，但该反应仍会导致一些DNA结构损伤（单链DNA断裂）。
