Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus

被引:42
作者
Urvil, PT
Kakiuchi, N
Zhou, DM
Shimotohno, K
Kumar, PKR
Nishikawa, S
机构
[1] MINIST INT TRADE & IND,AGCY IND SCI & TECHNOL,NATL INST BIOSCI & HUMAN TECHNOL,TSUKUBA,IBARAKI 305,JAPAN
[2] KYOTO UNIV,INST VIRUS RES,KYOTO 606,JAPAN
[3] UNIV TSUKUBA,TSUKUBA,IBARAKI 305,JAPAN
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1997年 / 248卷 / 01期
关键词
hepatitis C virus; NS3; protein; in vitro selection; aptamer; RNA;
D O I
10.1111/j.1432-1033.1997.t01-1-00130.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic-selection strategy to isolate high-affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12-18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G-1, was found predominantly (71%) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate-modification-interference analysis we showed that the phosphate residues that are critical for the binding of 10G-1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28-U34 and A47-A55. The NS3-binding region in 10G-1 can serve as a basis for designing more potential inhibitors of the NS3 protein.
引用
收藏
页码:130 / 138
页数:9
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