Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro
Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro
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DOI：
10.1016/j.bbrc.2010.01.100
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发表时间：
2010-02-26
影响因子：
3.1
通讯作者：
Noda, Naohiro
中科院分区：
文献类型：
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作者：
Tani, Hidenori;Fujita, Osamu;Noda, Naohiro
我们已经开发了一种基于荧光共振能量转移（FRET）的连续荧光检测方法，用于体外RNA解旋酶活性的监测。使用丙型肝炎病毒（HCV）NS3解旋酶作为模型检测该检测试剂盒。我们制备了双链RNA（dsRNA）底物，其具有与3'猝灭剂标记的链杂交的5'荧光团标记的链。当dsRNA被解旋酶解旋时，荧光团的荧光在链分离后发射。与传统的基于凝胶的测定不同，这种新的测定消除了复杂和耗时的步骤，并且可以用于简单地测量单个解旋酶反应中的实时动力学。我们的研究结果表明，Alexa Fluor 488和BHQ 1是一个有效的荧光团猝灭剂对，这种方法适用于定量测量HCV NS3的RNA解旋酶活性。此外，我们发现，几种海洋生物提取物对HCV NS3的RNA和DNA解旋酶活性表现出不同的抑制作用。我们建议，这种方法将是有用的，用于监测详细的动力学的RNA解旋机制和筛选RNA解旋酶抑制剂在高通量。(C)2010年爱思唯尔公司All rights reserved.
We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput. (C) 2010 Elsevier Inc. All rights reserved.