DEVELOPMENT OF NEW RNAAPTAMERS AGAINST HCV

抗 HCV 的新型 RNA 适体的开发

基本信息

批准号：
15590108
负责人：
NISHIKAWA Satoshi
金额：
$ 1.98万
依托单位：
NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590108/
关键词：
APTAMER HCV NS3 PROTEASE NS3 HELICASE IRES FRET RIBOZYME アプタマー

项目摘要

Infecting 3% of the world's population, Hepatitis C Virus(HCV) is the major infectious agent leading to non-A and non-B hepatitis. Non-structural protein 3(NS3) of HCV has two distinct activities, protease and helicase, which are essential for HCV proliferation. The translation of HCV starts at the internal ribosome entry site(IRES) and IRES is well conserved in HCV strains. Accordingly these proteins and RNA site are all good targets of anti-HCV drugs. In vitro selection, namely, SELEX(systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating APTMERs that have high affinity for target molecules from a randomized oligonucleotide pool. In order to develop new drug-lead compounds to block HCV, we did selection and obtained RNA aptamers specific to NS3 protease, NS3 helicase and HCV IRES, respectively. In the first step we established an assay system of anti-NS3 protease aptamer in cultured cells. Peptide substrate bearing the sequence of NS3 protease cleavage site sandwiched with fluorescent proteins CFP and YEP. In the next step we constructed efficient aptamer expression vector in cells by connecting cis-acting ribozyme and the aptamer. Efficient protease inhibition was detected by repeating this ribozyme-aptamer unit. We also rationally designed dual-functional aptamers by fusing anti-protease and anti-helicase aptamers. By conjugation of two different aptamers, both inhibition ability increased, especially against helicase four to five-fold inhibition was detected. As a result of selection against IRES, we found domain II of IRES is important target site in HCV RNA genome.

丙型肝炎病毒（HCV）感染了3％的世界人口，是导致非A和非B肝炎的主要感染剂。 HCV的非结构蛋白3（NS3）具有两种不同的活性，蛋白酶和解旋酶，这对于HCV增殖至关重要。 HCV的翻译始于内部核糖体入口位点（IRES），IRES在HCV菌株中的保守良好。因此，这些蛋白质和RNA位点都是抗HCV药物的良好靶标。体外选择，即SELEX（通过指数富集对配体的系统演变）是一种有用的策略，用于隔离从随机寡核苷酸池中对靶分子具有高亲和力的插座。为了开发新的药物铅化合物以阻断HCV，我们进行了选择，并分别获得了针对NS3蛋白酶，NS3解旋酶和HCV IRES的RNA Aptamers。在第一步中，我们在培养的细胞中建立了抗NS3蛋白酶适体的测定系统。带有NS3蛋白酶裂解位点序列的肽底物，夹杂着荧光蛋白CFP和YEP。在下一步中，我们通过连接顺式作用核酶和适体在细胞中构建了有效的适应性表达载体。通过重复该核酶 - 调子单元检测到有效的蛋白酶抑制作用。我们还通过融合抗癌和抗螺旋酶适体来合理设计的双功能适体。通过结合两种不同的适体，抑制能力都会提高，尤其是对四到五倍抑制的解旋酶。由于针对IRES的选择，我们发现IRES的II域II是HCV RNA基因组中的重要目标位点。