RNA origami scaffolds as a cryo-EM tool for investigating aptamer-ligand binding of a Broccoli-Pepper FRET pair

By N. S. Vallina, Ewan K. S. McRae, B. K. Hansen, Adrien Boussebayle, E. Andersen. 2022

RNA nanotechnology uses motifs from nature as well as aptamers from in vitro selection to construct nanostructures and devices for applications in RNA medicine and synthetic biology. The RNA origami method allows cotranscriptional folding of large RNA scaffolds that can position functional motifs in a precise manner, which has been verified by Förster Resonance Energy Transfer (FRET) between fluorescent aptamers. Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA nanostructures. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight. Here, we place aptamers on the RNA origami scaffolds to increase the contrast for cryo-EM and solve the structure of a new Broccoli-Pepper FRET pair. We identify different modes of ligand binding of the two aptamers and verify by selective probing. 3D variability analysis of the cryo-EM data show that the relative position between the two bound fluorophores on the origami fluctuate by only 3.5 Angstrom. Our results demonstrate the use of RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision. The Broccoli-Pepper apta-FRET pair has potential use for developing advanced sensors that are sensitive to small conformational changes.

Citations in the corpus

• Assessment of three-dimensional RNA structure prediction in CASP15 (2023, neutral): “Despite these caveats, the general global fold accuracy for RNA-only targets — even those without homologs of known structure — and the ability of models, with some curation, to enable molecular replacement of all 4 x-ray diffraction data sets suggest reason for optimism.”