Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro.
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.
Tani
Hidenori
H
Department of Life Science and Medical Bio-Science, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan.
Fujita
Osamu
O
Furuta
Atsushi
A
Matsuda
Yasuyoshi
Y
Miyata
Ryo
R
Akimitsu
Nobuyoshi
N
Tanaka
Junichi
J
Tsuneda
Satoshi
S
Sekiguchi
Yuji
Y
Noda
Naohiro
N
D000998:Antiviral Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology*; D020365:RNA Helicases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014020:Tissue Extracts / Q000737:chemistry / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D020365:RNA Helicases; D012330:RNA, Double-Stranded; D014020:Tissue Extracts; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases