RNA origami scaffolds facilitate cryo-EM characterization of a Broccoli–Pepper aptamer FRET pair

Abstract

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.

RNA target highlights in CASP15: Evaluation of predicted models by structure providers

“…In our opinion, synthetic nucleotide sequences represent a particularly interesting class of targets for structural prediction contests because they have very little sequence similarity to known RNA structures, forcing predictions to rely first on the principles of RNA folding rather than comparative modeling. Furthermore, the motifs that are incorporated from known structures and can be generated by comparative modeling (e.g., KLs and aptamers) are often different in the context of a larger RNA and in solution than they are in isolation in a crystal structure 45 …”

Assessment of three‐dimensional RNA structure prediction in CASP15

“…More specifically, we compared the following structure pairs in targets with multiple conformations (see also Table 1): the point-mutations for the CPEB3 ribozyme 61 (R1107 vs. R1108), the apo and holo structures of the aptamer Apta-FRET 62 scores were based on clashscore, 28 which has been used widely for both protein and RNA structural assessment. We used the following weighted sum of scores:…”

“…Interestingly, for the same R1138 six-helix-bundle, cryo-EM also captured a distinct "young" structure for the RNA (Figure 7J) that is dominant immediately after the transcription of the RNA and requires hours to resolve into the "mature" form. 62 The "young" and "mature" structures do not differ in their Watson-Crick-Franklin helices but, to interconvert, would require breaking of a kissing loop interaction, twisting of the two kissing elements about their helical axes, and then reformation of the kissing loop. 63 None of the CASP models produced models close to the "young" structure.…”

RNA tertiary structure prediction in CASP15 by the GeneSilico group: Folding simulations based on statistical potentials and spatial restraints

“…We manually adjusted the consensus secondary structure generated by MeSSPredRNA based on information from literature 34,35 and used it with RNAComposer to build an initial model. Using template-based modeling, we generated the 3D structures of the two aptamers (RCSB PDB entries of templates 5ob3 32 and 7eop 36 ) and manually inserted them into the full-length model.…”