Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro

Biochemical and Biophysical Research Communications
Hidenori TaniNaohiro Noda

Abstract

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.

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Citations

Oct 24, 2012·Journal of Biomedicine & Biotechnology·Hsin-Hsien Lin, Cheng-Yang Huang
Jun 20, 2014·Nature Protocols·Ali R ÖzeşChristopher S Fraser
Apr 5, 2014·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Kazi Abdus SalamNobuyoshi Akimitsu
Aug 12, 2015·International Journal of Molecular Sciences·Atsushi FurutaNaohiro Noda
Mar 15, 2015·Nucleic Acids Research·Oscar MendozaJean-Louis Mergny
Jul 8, 2015·Molecular and Cellular Biology·Haydar ÇelikAykut Üren
Aug 21, 2019·Wiley Interdisciplinary Reviews. RNA·Donny D LicatalosiEckhard Jankowsky
Feb 4, 2011·Chemical Society Reviews·Nan Dai, Eric T Kool
Mar 29, 2013·Journal of Biomolecular Screening·William R ShadrickDavid N Frick
Dec 22, 2016·The Journal of Biological Chemistry·Aditya MojumdarSilvia Onesti
Dec 30, 2017·Analytical Chemistry·Dimithree KahandaJason D Slinker

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