Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase
Selection of RNA aptamers that are specific and high-affinity ligands of the hepatitis C virus RNA-dependent RNA polymerase
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DOI：
10.1128/jvi.76.8.3688-3696.2002
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发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
Tomei, L
中科院分区：
文献类型：
--
作者：
Biroccio, A;Hamm, J;Tomei, L
为了寻找非结构性513（NS 5 B）聚合酶的特异性和高亲和力配体的小RNA分子，我们通过SELEX（通过指数扩增的配体系统进化）筛选具有携带C末端生物素化序列的NS 5 BDeltaC 55酶的结构限制的RNA文库。在所选的克隆中，两个适体似乎是NS 5 B的高亲和力配体，具有在低纳摩尔范围内的表观解离常数。它们共享一个可以呈现茎环结构的序列。通过突变分析，该结构已显示对应于负责与NS 5 B紧密相互作用的RNA基序。由于不能观察到与GB病毒B（GBV-B）NS 5 B蛋白的相互作用，适体似乎对丙型肝炎病毒（HCV）聚合酶具有高度特异性。这与HCV NS 5 B聚合酶的活性被所选适体有效抑制，而GBV-B和脊髓灰质炎病毒3D聚合酶均不受影响的观察结果一致。NS 5 B活性的抑制机制被证明是非竞争性的模板RNA，这表明适配体和模板RNA不结合到相同的网站。事实上，在拇指结构域的基本暴露表面中引入的突变严重损害了RNA适体的结合和活性抑制。
In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 513 (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5BDeltaC55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to he highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.