The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo . A detailed structure–function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

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Cryogenic electron microscopy (cryo-EM) is a promising method for characterizing the structure of larger RNA structures and complexes. However, the structure of individual aptamers is difficult to solve by cryo-EM due to their low molecular weight and a high signal-to-noise ratio. By placing RNA aptamers on larger RNA scaffolds, the contrast for cryo-EM can be increased to allow the determination of the tertiary structure of the aptamer. Here we use the RNA origami method to scaffold two fluorescent aptamers (Broccoli and Pepper) in close proximity and show that their cognate fluorophores serve as donor and acceptor for FRET. Next, we use cryo-EM to characterize the structure of the RNA origami with the two aptamers to a resolution of 4.4 Å. By characterizing the aptamers with and without ligand, we identify two distinct modes of ligand binding, which are further supported by selective chemical 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 Å. Our results demonstrate a general approach for using RNA origami scaffolds for characterizing small RNA motifs by cryo-EM and for positioning functional RNA motifs with high spatial precision.

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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.