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

17 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

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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