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.