Sensitive detection of endonuclease activity and inhibition using gold nanorods
Sensitive detection of endonuclease activity and inhibition using gold nanorods
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使用金纳米棒灵敏检测核酸内切酶活性和抑制
DOI：
10.1016/j.bios.2012.01.034
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发表时间：
2012-04-15
影响因子：
12.6
通讯作者：
Zhang, Chengxiao
中科院分区：
文献类型：
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作者：
Deng, Jing;Jin, Yan;Zhang, Chengxiao
因此，建立可靠、灵敏的核酸酶活性测定方法具有重要意义。考虑到这一目标，我们报告了一种新的策略，核酸酶检测利用高效的荧光共振能量转移（FRET）之间的金纳米棒（GNRs）和荧光素标记的单链DNA（FDNA）。在与GNR混合时，带正电荷的GNR和带负电荷的FDNA之间的FRET引起FDNA的荧光降低。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，荧光恢复时，FDNA 1/cDNA 1杂交被EcoRI内切酶切割成小片段，导致FRET效率下降，因为GNRs和缩短的DNA片段之间的静电相互作用减弱。通过荧光检测技术实时研究了EcoRI酶的活性随作用时间的变化。在优化的条件下，酶切组分与EcoRI浓度在1.0 × 10（-3）~ 1.0 × 10（-1）U μ L-1范围内呈线性关系，检测限为6.5 × 10（-4）U μ L-1，比以前的报道好得多或至少相当。凝胶电泳、荧光各向异性和透射电镜分析也证实了EcoRI内切酶对DNA的位点特异性切割，表明该方法是研究序列特异性蛋白质-DNA相互作用的一种可行和合理的方法。BamH Ⅰ酶活测定法是一种较为普遍应用的研究核酸内切酶活性的方法。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核酸内切酶抑制剂。因此，FRET技术是一种简便、灵敏、有效的研究核酸内切酶活性和抑制作用的方法，有望为核酸酶抑制剂的筛选提供一种可行的方法。(C)2012 Elsevier B. V.保留所有权利。
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 x 10(-3) to 1.0 x 10(-1) U mu L-1, with a limit of detection of 6.5 x 10(-4) U mu L-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. (C) 2012 Elsevier B.V. All rights reserved.