Branched kissing loops for the construction of diverse RNA homooligomeric nanostructures.

In biological systems, large and complex structures are often assembled from multiple simpler identical subunits. This strategy-homooligomerization-allows efficient genetic encoding of structures and avoids the need to control the stoichiometry of multiple distinct units. It also allows the minimal number of distinct subunits when designing artificial nucleic acid structures. Here, we present a robust self-assembly system in which homooligomerizable tiles are formed from intramolecularly folded RNA single strands. Tiles are linked through an artificially designed branched kissing-loop motif, involving Watson-Crick base pairing between the single-stranded regions of a bulged helix and a hairpin loop. By adjusting the tile geometry to gain control over the curvature, torsion and the number of helices, we have constructed 16 different linear and circular structures, including a finite-sized three-dimensional cage. We further demonstrate cotranscriptional self-assembly of tiles based on branched kissing loops, and show that tiles inserted into a transfer RNA scaffold can be overexpressed in bacterial cells.

Computer-Aided Design of RNA Origami Structures.

RNA nanostructures can be used as scaffolds to organize, combine, and control molecular functionalities, with great potential for applications in nanomedicine and synthetic biology. The single-stranded RNA origami method allows RNA nanostructures to be folded as they are transcribed by the RNA polymerase. RNA origami structures provide a stable framework that can be decorated with functional RNA elements such as riboswitches, ribozymes, interaction sites, and aptamers for binding small molecules or protein targets. The rich library of RNA structural and functional elements combined with the possibility to attach proteins through aptamer-based binding creates virtually limitless possibilities for constructing advanced RNA-based nanodevices.In this chapter we provide a detailed protocol for the single-stranded RNA origami design method using a simple 2-helix tall structure as an example. The first step involves 3D modeling of a double-crossover between two RNA double helices, followed by decoration with tertiary motifs. The second step deals with the construction of a 2D blueprint describing the secondary structure and sequence constraints that serves as the input for computer programs. In the third step, computer programs are used to design RNA sequences that are compatible with the structure, and the resulting outputs are evaluated and converted into DNA sequences to order.