A novel high throughput screening assay for HCVNS3 helicase activity
A novel high throughput screening assay for HCVNS3 helicase activity
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DOI：
10.1016/s0166-3542(00)00085-1
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发表时间：
2000-06-01
影响因子：
7.6
通讯作者：
Bedard, J
中科院分区：
文献类型：
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作者：
Alaoui-Ismaili, MH;Gervais, C;Bedard, J
采用Flashplate（TM）技术建立了一种检测丙型肝炎病毒（HCV）NS3解旋酶活性的新方法。本试验涉及使用DNA双链体底物和大肠杆菌中产生的重组HCV NS3。DNA双链体由一对寡核苷酸组成，一个生物素化，另一个在其各自的5'末端放射性标记。将该DNA双链体固定化。通过生物素分子，在中性抗生物素蛋白包被的SMP103FlashplateTM（NENLifeScienceProducts）的表面上。解旋酶活性导致放射性标记的寡核苷酸的释放，其相对于对照威尔斯孔转化为信号降低。HCV NS3解旋酶活性的生物化学表征使用该测定进行。我们证明了NS3介导的解旋与孔中DNA底物的量和反应中NS3的浓度都成比例。大多数NS3介导的解旋在孵育的最初60分钟内实现。正如预期的反应是ATP依赖性的，并发现受MgCl2，MnCl2，KCl，EDTA的浓度和pH值的影响。我们发现这种测定是高度可重复的，因为只有轻微的变化时，观察到共68个解旋酶反应在一个板上进行。因此，该Flashplate（TM）解旋酶测定是快速、方便和可重复的。这些标准使其适合于潜在的NS3解旋酶抑制剂的高通量筛选。(C)2000 Elsevier Science B.V.保留所有权利。
A novel assay for measurement of Hepatitis C virus (HCV) NS3 helicase activity was developed using Flashplate(TM) technology. This assay involves the use of a DNA duplex substrate and recombinant HCV NS3 produced in Escherichia coli. The DNA duplex consisted of a pair of oligonucleotides, one biotinylated, the other radiolabeled at their respective 5' termini. This DNA duplex was immobilized. via the biotin molecule, on the surface of a neutravidin-coated SMP103 Flashplate(TM) (NEN Life Science Products). Helicase activity results in the release of the radiolabeled oligonucleotide, which translates in signal reduction with respect to control wells. Biochemical characterization of the HCV NS3 helicase activity was performed using this assay. We demonstrated that the NS3-mediated unwinding is proportional to both the amount of DNA substrate in the well, and to the NS3 concentration in the reaction. Most of the NS3-mediated unwinding was achieved in the initial 60 min of incubation. As expected the reactions were ATP-dependent and found to be affected by the concentration of MgCl2, MnCl2, KCl, EDTA, and by pH. We found this assay to be highly reproducible since only slight variation was observed when a total of 68 helicase reactions were performed on one plate. Therefore, this Flashplate(TM) helicase assay is fast, convenient and reproducible. These criteria make it suitable for high throughput screening of potential NS3 helicase inhibitors. (C) 2000 Elsevier Science B.V. All rights reserved.