Out-of-Plane Aptamer Functionalization of RNA Three-Helix Tiles

Co-transcriptionally folding RNA nanostructures have great potential as biomolecular scaffolds, which can be used to organize small molecules or proteins into spatially ordered assemblies. Here, we develop an RNA tile composed of three parallel RNA double helices, which can associate into small hexagonal assemblies via kissing loop interactions between its two outer helices. The inner RNA helix is modified with an RNA motif found in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV), which provides a 90° bend. This modification is used to functionalize the RNA structures with aptamers pointing perpendicularly away from the tile plane. We demonstrate modifications with the fluorogenic malachite green and Spinach aptamers as well with the protein-binding PP7 and streptavidin aptamers. The modified structures retain the ability to associate into larger assemblies, representing a step towards RNA hybrid nanostructures extending in three dimensions.