Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an, China.
Biosens Bioelectron. 2012 Apr 15;34(1):144-50. doi: 10.1016/j.bios.2012.01.034. Epub 2012 Feb 9.
It is important to develop reliable and sensitive methods for assay of nuclease activity. With this goal in mind, we report a new strategy for nuclease assay by taking advantage of efficient fluorescence resonance energy transfer (FRET) between gold nanorods (GNRs) and fluorescein-tagged single-stranded DNA (FDNA). Upon mixing with GNRs, the FRET between positively charged GNRs and negatively charged FDNA caused a decrease in fluorescence of FDNA. The formation of FDNA/cDNA duplex further improved the FRET efficiency, leading to a significant decrease in fluorescence intensity. However, fluorescence is restored when FDNA1/cDNA1 hybrid was cleaved into small fragments by EcoRI endonucleases, resulting in a decrease in FRET efficiency because of weakened electrostatic interaction between GNRs and the shortened DNA fragments. Activity of EcoRI endonuclease has been real-time studied by monitoring fluorescence change with the prolonging of interaction time. Under optimized conditions, the cleaved fraction is linear with EcoRI concentration over the range of 1.0×10(-3) to 1.0×10(-1) U μL(-1), with a limit of detection of 6.5×10(-4) U μL(-1) which is much better or at least comparable to previous reports. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis, fluorescence anisotropy and TEM analysis, which indicated that this method is a feasible and reasonable approach to study sequence-specific protein-DNA interactions. Assay of BamHI activity demonstrated that it is a more universally applied method for studying the activity of endonuclease. Furthermore, this fluorescence assay has been also used for studying the inhibition of EcoRI endonuclease activity. Importantly, experimental results suggested that endonuclease inhibitors can be screened by monitoring the change of fluorescence change. Therefore, this FRET assay is a simple, sensitive and effective approach to study endonuclease activity and inhibition, and as such, it promises to provide a feasible method to screen nuclease inhibitors.
开发用于测定核酸酶活性的可靠且灵敏的方法非常重要。基于这一目标，我们报道了一种利用金纳米棒（GNRs）和荧光素标记的单链 DNA（FDNA）之间有效的荧光共振能量转移（FRET）来测定核酸酶活性的新策略。与 GNRs 混合后，带正电荷的 GNRs 和带负电荷的 FDNA 之间的 FRET 导致 FDNA 的荧光强度降低。FDNA/cDNA 双链体的形成进一步提高了 FRET 效率，导致荧光强度显著降低。然而，当 FDNA1/cDNA1 杂交物被 EcoRI 内切酶切割成小片段时，荧光恢复，导致由于 GNRs 与缩短的 DNA 片段之间的静电相互作用减弱，FRET 效率降低。通过监测相互作用时间延长时荧光变化，实时研究了 EcoRI 内切酶的活性。在优化条件下，随着 EcoRI 浓度在 1.0×10(-3) 至 1.0×10(-1) U μL(-1)范围内的增加，切割部分呈线性关系，检测限为 6.5×10(-4) U μL(-1)，优于或至少与以前的报道相当。EcoRI 内切酶的位点特异性 DNA 切割也通过凝胶电泳、荧光各向异性和 TEM 分析得到了验证，这表明该方法是研究序列特异性蛋白-DNA 相互作用的一种可行且合理的方法。BamHI 活性测定表明，该方法更普遍适用于研究内切酶的活性。此外，该荧光测定法还用于研究 EcoRI 内切酶活性的抑制。重要的是，实验结果表明，可以通过监测荧光变化来筛选内切酶抑制剂。因此，这种 FRET 测定法是研究内切酶活性和抑制的一种简单、灵敏且有效的方法，有望为筛选核酸酶抑制剂提供一种可行的方法。
