针对丙型肝炎病毒NS3解旋酶结构域的RNA适体的体外筛选。

In vitro selection of RNA aptamers against the HCV NS3 helicase domain.

作者信息

Nishikawa Fumiko, Funaji Kohei, Fukuda Kotaro, Nishikawa Satoshi

机构信息

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

出版信息

Oligonucleotides. 2004;14(2):114-29. doi: 10.1089/1545457041526335.

DOI:10.1089/1545457041526335

PMID:15294075

Abstract

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct domains, protease and helicase, that are essential for HCV proliferation. Therefore, NS3 is considered a target for anti-HCV treatment. To study RNA aptamers of the NS3 helicase domain, we carried out in vitro selection against the HCV NS3 helicase domain. RNA aptamers obtained after eight generations possessed 5' extended single-stranded regions and the conserved sequence (5'-GGA(U/C)GGAGCC-3') at stem-loop regions. Aptamer 5 showed strong inhibition of helicase activity in vitro. Deletion and mutagenesis analysis clarified that the conserved stem-loop is important and that the whole structure is needed for helicase inhibition. We compared the inhibition of helicase activity between aptamer 5 and 3'+-UTR of HCV.

摘要

丙型肝炎病毒（HCV）的非结构蛋白3（NS3）有两个不同的结构域，即蛋白酶和螺旋酶，它们对HCV的增殖至关重要。因此，NS3被认为是抗HCV治疗的一个靶点。为了研究NS3螺旋酶结构域的RNA适配体，我们针对HCV NS3螺旋酶结构域进行了体外筛选。经过八轮筛选得到的RNA适配体在茎环区域具有5'延伸的单链区域和保守序列（5'-GGA(U/C)GGAGCC-3'）。适配体5在体外对螺旋酶活性表现出强烈的抑制作用。缺失和诱变分析表明，保守的茎环结构很重要，并且整个结构对于螺旋酶抑制是必需的。我们比较了适配体5和HCV 3'+-UTR对螺旋酶活性的抑制作用。