Switchback RNA

Abstract

Intricately designed DNA and RNA motifs guide the assembly of robust and functional nucleic acid nanostructures. In this work, we present a globally left-handed RNA motif with two parallel strands called switchback RNA and report its assembly, biophysical, and biochemical characterization. Switchback RNA can be assembled in buffers without Mg²⁺, with improved thermal stability in buffers containing Mg²⁺, Na⁺, or K⁺. Differences in the binding of small molecules to switchback RNA and conventional RNA indicate design-based approaches for small molecule loading on RNA nanostructures. Further, the differential affinity of the two component strands in switchback or conventional duplex conformations allows for toehold-less strand displacement. Enzyme studies showed that the switchback and conventional RNA structures have similar levels of nuclease resistance. These results provide insights for employing switchback RNA as a structural motif in RNA nanotechnology. Our observation that RNA strands with switchback complementarity can form stable complexes at low magnesium concentrations encourages studies into the potential occurrence of switchback RNA in nature.

Figure 1

Design and assembly of switchback RNA. (a) Schematic representation of switchback RNA and conventional RNA duplex. (b) Nondenaturing gel image showing the formation of switchback RNA in Mg²⁺-free TAE buffer. (c) Comparison of the UV-thermal melting curves of switchback RNA and conventional RNA duplex in TAE buffer. (d) Effect of Mg²⁺ on the melting temperature of switchback RNA. (e) Comparison of the circular dichroism (CD) spectra of switchback RNA and conventional RNA duplex in TAE buffer. (f) Mg²⁺ concentration-dependent change in the CD spectra of switchback RNA.

Figure 2

Strand competition and displacement between switchback RNA and conventional duplex. (a) Competition between switchback complement (rY) and a duplex complement (rZ_L) to bind to rX. (b) A duplex complement (rZ_L) displaces a switchback complement (rY) from a preassembled switchback RNA. (c) Addition of switchback complement (rY) to a preformed duplex does not displace the duplex complement.

Figure 3

Small molecule binding to switchback RNA. (a) Chemical structure of ethidium bromide. (b) Fluorescence enhancement of ethidium bromide when bound to switchback RNA and conventional duplex. (c) Effect of Mg²⁺ on the fluorescence of ethidium bromide bound to switchback RNA and conventional duplex. (d) Chemical structure of thiazole orange. (b) Fluorescence enhancement of thiazole orange when bound to switchback RNA and conventional duplex. (c) Effect of Mg²⁺ on the fluorescence of thiazole orange bound to switchback RNA and conventional duplex.

Figure 4

Nuclease resistance of switchback RNA. (a) Degradation of switchback RNA and conventional duplex when treated with different amounts of RNase III. (b) Degradation of switchback RNA:DNA hybrid and conventional hybrid when treated with different amounts of RNase H.