Urvil P T, Kakiuchi N, Zhou D M, Shimotohno K, Kumar P K, Nishikawa S
National Institute of Bioscience and Human Technology, AIST, Tsukuba Science City, Ibaraki, Japan.
Eur J Biochem. 1997 Aug 15;248(1):130-8. doi: 10.1111/j.1432-1033.1997.t01-1-00130.x.
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.
人类丙型肝炎病毒（HCV）的RNA基因组被翻译为一个大的前体多聚蛋白。HCV的NS3蛋白酶在将多聚蛋白加工成功能性病毒蛋白的过程中起着关键作用。我们采用了一种体外遗传选择策略，以分离与NS3蛋白，特别是其蛋白酶结构域结合的高亲和力RNA适体。从一个具有12 - 18个核苷酸随机序列核心的RNA文库开始，经过10轮选择和扩增后，筛选出了与NS3蛋白特异性结合的适体。在筛选出的文库中，主要发现了一种单一适体10G - 1（占71%）。这种适体可以以650 nM的结合常数与NS3蛋白结合，并在体外抑制其蛋白水解活性。通过磷酸修饰干扰分析，我们表明，对10G - 1与NS3结合至关重要的磷酸残基位于适体的选定区域内，并且结合涉及与G28 - U34和A47 - A55区域的磷酸基团的静电接触。10G - 1中的NS3结合区域可作为设计更多潜在NS3蛋白抑制剂的基础。
