Synthesizing topological structures containing RNA.
Synthesizing topological structures containing RNA.
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DOI：
10.1038/ncomms14936
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发表时间：
2017-03-31
影响因子：
16.6
通讯作者：
Weizmann Y
中科院分区：
文献类型：
--
作者：
Liu D;Shao Y;Chen G;Tse-Dinh YC;Piccirilli JA;Weizmann Y
尽管在 DNA 和蛋白质中观察到打结和缠结，但它们在 RNA 中的存在仍然是一个谜。合成 RNA 拓扑结构对于理解与 RNA 拓扑相关的物理和生物特性具有重要意义，而这些特性反过来又可以促进识别自然存在的拓扑非平凡 RNA 分子。在这里，我们展示了包含无强碱基配对相互作用的单链RNA（ssRNA）的拓扑结构可以通过配置RNA-DNA混合四路连接或通过单链DNA（ssDNA）拓扑结构的模板引导合成来创建。通过使用构建的ssRNA结作为高灵敏度拓扑探针，我们发现大肠杆菌DNA拓扑异构酶I具有较低的RNA拓扑异构酶活性，并且R173A点突变消除了ssRNA的解结活性，但不消除ssDNA的解结活性。此外，我们发现了逆转录（RT）的拓扑抑制，并获得了相同序列的 ssRNA 结和环的不同 RT-PCR 模式。 在体内，复杂的拓扑结构已在蛋白质和 DNA 中被发现，但它们在 RNA 中的存在仍不清楚。在这里，作者设计了包含单链 RNA 的合成拓扑结构，为研究有关 RNA 拓扑的生物学相关问题提供了工具。
Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. Synthetic RNA topological structures are significant for understanding the physical and biological properties pertaining to RNA topology, and these properties in turn could facilitate identifying naturally occurring topologically nontrivial RNA molecules. Here we show that topological structures containing single-stranded RNA (ssRNA) free of strong base pairing interactions can be created either by configuring RNA–DNA hybrid four-way junctions or by template-directed synthesis with a single-stranded DNA (ssDNA) topological structure. By using a constructed ssRNA knot as a highly sensitive topological probe, we find that Escherichia coli DNA topoisomerase I has low RNA topoisomerase activity and that the R173A point mutation abolishes the unknotting activity for ssRNA, but not for ssDNA. Furthermore, we discover the topological inhibition of reverse transcription (RT) and obtain different RT–PCR patterns for an ssRNA knot and circle of the same sequence. In vivo, complex topologies have been identified in proteins and DNA, while their existence in RNA is still unclear. Here, the authors design synthetic topological structures containing single stranded RNA, offering tools for investigating biologically relevant questions about RNA topology.