Nanopore-based conformational analysis of a viral RNA drug target.
Nanopore-based conformational analysis of a viral RNA drug target.
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DOI：
10.1021/nn501969r
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发表时间：
2014-06-24
期刊：
影响因子：
17.1
通讯作者：
Wanunu M
中科院分区：
文献类型：
--
作者：
Shasha C;Henley RY;Stoloff DH;Rynearson KD;Hermann T;Wanunu M
纳米孔是单分子传感器，通过实现少量样品的无标记检测，显示出作为生物分子分析工具的特殊前景。在本文中，我们证明了纳米孔能够检测抗病毒RNA药物靶标的构象。丙型肝炎病毒使用内部核糖体进入位点（IRES）基序，以便通过对接到其宿主细胞中的核糖体来启动翻译。因此，IRES是一个可行的和重要的药物靶标。药物诱导的HCV IRES基序构象的变化，从弯曲到直的构象，已经显示出抑制HCV复制。然而，目前还没有直接的方法来分析候选小分子药物对RNA构象的影响。在本文中，我们表明，通过3 nm直径的纳米孔的RNA易位动力学是构象敏感的，通过展示短病毒RNA基序的弯曲和直的构象之间的运输时间的差异。检测是可能的，因为弯曲的RNA在3 nm孔中停滞，导致比直RNA更长的分子停留时间。对照实验表明，较弱的药物的结合不产生构象变化，与独立的荧光测量一致。因此，RNA构象的纳米孔测量可用于探测各种RNA基序的结构，以及小分子结合后RNA的结构变化。
Nanopores are single-molecule sensors that show exceptional promise as a biomolecular analysis tool by enabling label-free detection of small amounts of sample. In this paper, we demonstrate that nanopores are capable of detecting the conformation of an antiviral RNA drug target. The hepatitis C virus uses an internal ribosome entry site (IRES) motif in order to initiate translation by docking to ribosomes in its host cell. The IRES is therefore a viable and important drug target. Drug-induced changes to the conformation of the HCV IRES motif, from a bent to a straight conformation, have been shown to inhibit HCV replication. However, there is presently no straightforward method to analyze the effect of candidate small-molecule drugs on the RNA conformation. In this paper, we show that RNA translocation dynamics through a 3 nm diameter nanopore is conformation-sensitive by demonstrating a difference in transport times between bent and straight conformations of a short viral RNA motif. Detection is possible because bent RNA is stalled in the 3 nm pore, resulting in longer molecular dwell times than straight RNA. Control experiments show that binding of a weaker drug does not produce a conformational change, as consistent with independent fluorescence measurements. Nanopore measurements of RNA conformation can thus be useful for probing the structure of various RNA motifs, as well as structural changes to the RNA upon small-molecule binding.