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

Nestor Sampedro Vallina, Ewan K.S. McRae, Bente Kring Hansen, Adrien Boussebayle, Ebbe Sloth Andersen

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)4613-4624
Number of pages12
JournalNucleic Acids Research
Issue number9
StatePublished - May 22 2023


  • Aptamers, Nucleotide/chemistry
  • Cryoelectron Microscopy/methods
  • Fluorescence Resonance Energy Transfer/methods
  • Ligands
  • RNA/chemistry
  • Nucleic Acid Conformation

ASJC Scopus subject areas

  • Genetics


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