High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

Hidenori Tani, Nobuyoshi Akimitsu, Osamu Fujita, Yasuyoshi Matsuda, Ryo Miyata, Satoshi Tsuneda, Masayuki Igarashi, Yuji Sekiguchi, Naohiro Noda

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    Abstract

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5′-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3′-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

    Original languageEnglish
    Pages (from-to)1054-1059
    Number of pages6
    JournalBiochemical and Biophysical Research Communications
    Volume379
    Issue number4
    DOIs
    Publication statusPublished - 2009 Feb 20

    Fingerprint

    High-Throughput Screening Assays
    Guanine
    Viruses
    Hepacivirus
    Quenching
    Assays
    Screening
    Fluorescence
    Throughput
    Cell Extracts
    Fluorescent Dyes
    Proteins
    Coloring Agents
    Microorganisms
    Electrons
    DNA

    Keywords

    • BODIPY FL
    • Fluorescence quenching
    • Hepatitis C virus
    • NS3 helicase
    • Photoinduced electron transfer

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Cell Biology
    • Molecular Biology

    Cite this

    High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon. / Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro.

    In: Biochemical and Biophysical Research Communications, Vol. 379, No. 4, 20.02.2009, p. 1054-1059.

    Research output: Contribution to journalArticle

    Tani, Hidenori ; Akimitsu, Nobuyoshi ; Fujita, Osamu ; Matsuda, Yasuyoshi ; Miyata, Ryo ; Tsuneda, Satoshi ; Igarashi, Masayuki ; Sekiguchi, Yuji ; Noda, Naohiro. / High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon. In: Biochemical and Biophysical Research Communications. 2009 ; Vol. 379, No. 4. pp. 1054-1059.
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    AU - Miyata, Ryo

    AU - Tsuneda, Satoshi

    AU - Igarashi, Masayuki

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