Design, assembly, and evaluation of RNA-protein nanostructures

The use of RNA-protein interaction motifs (RNP motifs) to design and build nanoscale objects has the potential to expand the field of RNA nanotechnology. In principle, RNP motifs can be integrated easily into RNA nano objects, providing an alternative technique to increase the functional and structural complexities of the RNA. Investigating the design principles of RNP nanostructures will enable the construction of highly sophisticated biomacromolecular complexes such as ribosomes from scratch. As an initial step towards this goal, we designed and constructed triangular-like nanostructures by employing box C/D kink-turn (K-turn)-L7Ae RNP motifs. We showed that the K-turn RNA and the ribosomal protein L7Ae could form a nanostructure shaped like an equilateral triangle that consists of the three proteins attached to the tips of the RNA scaffold. The construction of the complex depends on L7Ae binding to the K-turn motifs in the RNA. The RNP motif allows the RNA to bend by approximately 60° at three positions to form a nanoscale triangle. Functional RNP triangles with desired protein modules at the three tips can be constructed in a modular manner. Here, we describe how to design, construct, and evaluate the RNP nanostructures.