Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease

被引:84
作者
Fukuda, K
Vishnuvardhan, D
Sekiya, S
Hwang, J
Kakiuchi, N
Kazunari, T
Shimotohno, K
Kumar, PKR
Nishikawa, S [1 ]
机构
[1] MITI, AIST, Natl Inst Biosci & Human Technol, Tsukuba Sci City 3058566, Japan
[2] Univ Tsukuba, Inst Appl Biochem, Tsukuba Sci City, Japan
[3] MITI, AIST, Natl Inst Adv Interdisciplinary Res, Tsukuba Sci City, Japan
[4] Kyoto Univ, Inst Virus Res, Kyoto, Japan
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 12期
关键词
HCV; RNA aptamer; SELEX; NS3 serine protease;
D O I
10.1046/j.1432-1327.2000.01400.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
Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide Delta NS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of Delta NS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to Delta NS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of Delta NS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for Delta NS3 with a K-i approximate to 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.
引用
收藏
页码:3685 / 3694
页数:10
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