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

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.

journal_name

Biochem Biophys Res Commun

journal_title

Biochemical and biophysical research communications

authors

Tani H,Fujita O,Furuta A,Matsuda Y,Miyata R,Akimitsu N,Tanaka J,Tsuneda S,Sekiguchi Y,Noda N

doi

10.1016/j.bbrc.2010.01.100

subject

Has Abstract

pub_date

2010-02-26 00:00:00

pages

131-6

issue

1

eissn

0006-291X

issn

1090-2104

pii

S0006-291X(10)00149-X

journal_volume

393

pub_type

杂志文章

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