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 language | English |
|---|---|
| Pages (from-to) | 1054-1059 |
| Number of pages | 6 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 379 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2009 Feb 20 |
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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 journal › Article
}
TY - JOUR
T1 - High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon
AU - Tani, Hidenori
AU - Akimitsu, Nobuyoshi
AU - Fujita, Osamu
AU - Matsuda, Yasuyoshi
AU - Miyata, Ryo
AU - Tsuneda, Satoshi
AU - Igarashi, Masayuki
AU - Sekiguchi, Yuji
AU - Noda, Naohiro
PY - 2009/2/20
Y1 - 2009/2/20
N2 - 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.
AB - 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.
KW - BODIPY FL
KW - Fluorescence quenching
KW - Hepatitis C virus
KW - NS3 helicase
KW - Photoinduced electron transfer
UR - http://www.scopus.com/inward/record.url?scp=58949094336&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=58949094336&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2009.01.020
DO - 10.1016/j.bbrc.2009.01.020
M3 - Article
C2 - 19150342
AN - SCOPUS:58949094336
VL - 379
SP - 1054
EP - 1059
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -