Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Nat Commun. 2017 Mar 31;8:14936. doi: 10.1038/ncomms14936.
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.
尽管在 DNA 和蛋白质中已经观察到了打结和缠绕现象，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 拓扑结构相关的物理和生物学特性具有重要意义，而这些特性反过来又有助于识别自然存在的具有非平凡拓扑结构的 RNA 分子。在这里，我们表明，通过配置 RNA-DNA 杂交四链结或使用单链 DNA（ssDNA）拓扑结构进行模板指导合成，可以创建不含强碱基配对相互作用的单链 RNA（ssRNA）的拓扑结构。通过使用构建的 ssRNA 结作为高度敏感的拓扑探针，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，但对 ssDNA 没有影响。此外，我们发现逆转录（RT）的拓扑抑制作用，并获得了相同序列的 ssRNA 结和环的不同 RT-PCR 模式。
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