RNA origami scaffolds facilitate cryo-EM characterization of a Broccoli–Pepper aptamer FRET pair
By Néstor Sampedro Vallina, Ewan K. S. McRae, B. K. Hansen, Adrien Boussebayle, E. Andersen. 2023
34 citations recorded by the source corpus.
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.
Subfields: Atomic and Molecular Physics, and Optics, Molecular Biology, Structural Biology
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In Molecular Biology
- Andriy Kryshtafovych
- Daniel J. Rigden
- Rhiju Das
- Rachael Kretsch
- Thomas Mulvaney
- J. Beton
- Seyed Naeim Moafinejad
- Sunandan Mukherjee
In Structural Biology
Citations in the corpus
- Assessment of three-dimensional RNA structure prediction in CASP15 (2023, neutral): “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.”
- RNA tertiary structure prediction in CASP15 by the GeneSilico group: Folding simulations based on statistical potentials and spatial restraints (2023, methodology): “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.”
- CASP15 cryo-EM protein and RNA targets: refinement and analysis using experimental maps (2023, background): “R1126 a designed “ Traptamer ” at 5.6 Å resolution The refined predictions of the designed RNA target R1126, a designed RNA origami scaffold for a Broccoli and Pepper aptamer pair, 31 had lower average SMOC scores than the reference target structure.”