Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
Biochem Biophys Res Commun. 2010 Feb 26;393(1):131-6. doi: 10.1016/j.bbrc.2010.01.100. Epub 2010 Feb 1.
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.
我们开发了一种基于荧光共振能量转移（FRET）的连续荧光测定法，用于体外监测 RNA 解旋酶的活性。该测定法使用丙型肝炎病毒（HCV）NS3 解旋酶作为模型进行了测试。我们制备了带有 5'荧光标记链杂交到 3'淬灭标记链的双链 RNA（dsRNA）底物。当 dsRNA 被解旋酶解开时，荧光团的荧光在链分离后发出。与传统的基于凝胶的测定法不同，这种新的测定法消除了复杂且耗时的步骤，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。我们的结果表明，Alexa Fluor 488 和 BHQ1 是一种有效的荧光团-淬灭剂对，该测定法适用于 HCV NS3 的 RNA 解旋酶活性的定量测量。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。我们提出，该测定法将有助于监测 RNA 解旋机制的详细动力学，并进行高通量筛选 RNA 解旋酶抑制剂。
