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

Journal Article · Fri Feb 20 00:00:00 EST 2009 · Biochemical and Biophysical Research Communications

DOI:https://doi.org/10.1016/j.bbrc.2009.01.020· OSTI ID:21255898

Tani, Hidenori ^[1]; Akimitsu, Nobuyoshi ^[2]; Fujita, Osamu; Matsuda, Yasuyoshi ^[3]; Miyata, Ryo ^[3]; Tsuneda, Satoshi ^[1]; Igarashi, Masayuki ^[4]; Sekiguchi, Yuji ^[3]; Noda, Naohiro ^[3]

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1. Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480 (Japan)
2. Radioisotope Center, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan)
3. Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 (Japan)
4. Microbial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

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.

• OSTI ID: 21255898
• Journal Information: Biochemical and Biophysical Research Communications, Vol. 379, Issue 4; Other Information: DOI: 10.1016/j.bbrc.2009.01.020; PII: S0006-291X(09)00024-2; Copyright (c) 2009 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
DNA
DYES
ELECTRON TRANSFER
FLUORESCENCE
GUANINE
HEPATITIS
PROTEINS
QUENCHING
SCREENING
VIRUSES