Synthesizing topological structures containing RNA

Di Liu,Gang Chen,Yuk-Ching Tse-Dinh,Joseph A. Piccirilli,Yaming Shao,Yossi Weizmann

doi:10.1038/ncomms14936

Di Liu, Gang Chen + Show 4 more

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https://doi.org/10.1038/ncomms14936

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Abstract

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.

Highlights

Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma
This junction-based method can be further developed for synthetic RNA topologies by using the RNA–DNA hybrid 4WJ (Fig. 1c), which contains RNA helical strands and DNA crossover strands
RNA topological structures can be generated by folding RNA scaffolds into hybrid 4WJs with DNA staples

Summary

Introduction

Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. 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 singlestranded DNA (ssDNA) topological structure. Synthetic RNA topological structures can help us understand the physical and biological properties associated with RNA topology. Based on these properties, new tools and methods to identify the naturally occurring RNA topological structures can be developed. The resulting RNA topological structures are free of strong base-pairing interactions, enabling the RNA Topo activity study of E. coli DNA Topo I and the discovery of topological inhibition of reverse transcription (RT). We expect our work on synthetic RNA topological structures will stimulate research in the essentially unexplored area of RNA topology and RNA topoisomerase

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