Tani Hidenori, Akimitsu Nobuyoshi, Fujita Osamu, Matsuda Yasuyoshi, Miyata Ryo, Tsuneda Satoshi, Igarashi Masayuki, Sekiguchi Yuji, Noda Naohiro
Department of Life Science and Medical Bio-Science, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.
Biochem Biophys Res Commun. 2009 Feb 20;379(4):1054-9. doi: 10.1016/j.bbrc.2009.01.020. Epub 2009 Jan 14.
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.
我们利用荧光染料与鸟嘌呤碱基之间的光致电子转移导致的荧光猝灭现象，开发了一种新型的高通量筛选丙型肝炎病毒（HCV）非结构蛋白3（NS3）解旋酶抑制剂的检测方法。我们制备了双链DNA（dsDNA），其一条链的5'端用荧光染料（BODIPY FL）标记，并与互补链杂交，互补链的3'端含有鸟嘌呤碱基。当dsDNA被解旋酶解开时，染料由于从鸟嘌呤碱基上释放而发出荧光。我们的结果表明，该检测方法适用于HCV NS3解旋酶活性的定量检测，并且可用于高通量筛选抑制剂。此外，我们将该检测方法应用于从微生物细胞提取物中筛选NS3解旋酶抑制剂，发现了几种含有潜在抑制剂的细胞提取物。
