Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure.

Yosuke Takeuchi,Jean-Jacques Toulmé,Masayuki Endo,Eric Dausse,Guillaume Durand,Kumi Hidaka,Yuki Suzuki,Hiroshi Sugiyama

doi:10.1039/c5bm00274e

Abstract

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures.

Highlights

Structural diversity of RNA is one of the important properties of RNA molecules, which exhibit unique functions such as specific complex formation and catalysis
The results indicate that the cover strand reduced the ligand-independent binding between the GTPswitch and the Aptakiss
Value is close to that in the absence of guanosine triphosphate (GTP) (64.4%). These results indicate that GTP could selectively induce binding between the GTPswitch and the Aptakiss, whereas Adenosine triphosphate (ATP) could not (Fig. 3e and Table 2)

Summary

Introduction

Structural diversity of RNA is one of the important properties of RNA molecules, which exhibit unique functions such as specific complex formation and catalysis. We placed the GTPswitch and the target kissing loop Aptakiss into the cavity of a DNA frame and directly observed their interaction (Fig. 1a).[19–22] The GTPswitch was generated based on a KG51 RNA kissing hairpin, which can bind to the Aptakiss only in the presence of GTP.[16] We elongated the 5′ end of each RNA molecule and hybridized these molecules to the supporting DNA strands.

Results

Conclusion