RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.
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The sequences of the designs used and the images of the blueprints are available in the Supplementary Information. Text versions of the blueprints are available from the corresponding author upon request. The volumes from the final refinements of our cryo-EM single-particle analysis datasets have been deposited to the Electron Microscopy Data Bank (EMDB) under accession codes EMD-13633 (5HT-A), EMD-13926 (5HT-A-twist-corrected), EMD-13636 (5HT-B), EMD-13592 (5HT-B-3X), EMD-13627 (6HB), EMD-13628 (6HBC-young-1), EMD-13630 (6HBC-PBS-mature-1), EMD-13626 (6HBC-young-2) and EMD-13625 (6HBC-mature-2). The atomic models have been deposited to the Protein Data Bank (PDB) under the PDB codes 7PTQ (5HT-A), 7QDU (5HT-A-twist-corrected), 7PTS (5HT-B), 7PTK (6HBC-young-1) and 7PTL (6HBC-mature-1). The 16HS volumes reconstructed from the cryo-ET data have been deposited to the EMDB under accession codes EMD-28669 to EMD-28684.
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We dedicate our work to the late Ned Seeman, who pioneered the research field of structural nucleic acid nanotechnology and greatly inspired our work and directly or indirectly influenced our careers. We acknowledge the EMBION Cryo-EM Facility at iNANO, Aarhus University, for time under application ID 0042. We thank P. Nissen and A. Briegel for their early support of the project and valuable discussion, S. Sparvath for early design and experiments with the 6HB and A. Briegel and H. C. Høiberg for early NS-TEM characterization of 6HBs. We thank R. Rosendahl and C. Bus for technical assistance. The work at iNANO was supported by the Independent Research Fund Denmark under the Research Project 1 grant (9040-00425B) to E.S.A., the Canadian Natural Sciences and Engineering Research Council post-doctoral fellowship (532417) to E.K.S.M., the Carlsberg Foundation Research Infrastructure grant (CF20-0635) to E.S.A., the European Research Council (ERC) Consolidator grant (683305) to E.S.A. and Novo Nordisk Foundation Ascending Investigator grant (NNF20OC0060694) and Interdisciplinary Synergy grant (NNF21OC0070452) to E.S.A. The work at the Molecular Foundry, Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Basic Energy Sciences, of the US Department of Energy (contract no. DE-AC02-05CH11231) and the US National Institutes of Health (nos. R01HL115153, R01 GM104427, R01MH077303 and R01DK042667) to J.L. and G.R.
The authors declare no competing interests.
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Extended Data Fig. 1 Overview of RNA origami designs, cryo-EM reconstructions and atomic models used in the study.
a, Example cryo-EM reconstructions for sheets (top row) and bundles (bottom row). b, Table listing RNA origami designs by name, blueprint, number of nucleotides, EMD ID, Gold-Standard Fourier Shell Correlation (GSFSC) for the reconstructions, PDB ID and model resolution (see Supplementary Table 10 for further details).
Extended Data Fig. 2 Reconstruction and model measurements of 5HT.
a, 5HT-A reconstruction based on local refinement of the 3 central helices shown at contour levels 0.344, 0.267 and 0.136 from left to right and colour coded by local resolution. b, Curves+ analysis of the 5HT-A model with helical axis shown as black lines. c, Curves+ analysis with backbone shown as red lines with major and minor groove measurements shown as grey lines. d, Tabulated data from Curves+ analysis of the helical components from the 5HT-A model. *All helical regions exclude KLs. **Crossover values are only based on the three central helices. e, Structural comparison of 5HT-B and 5HT-B-V2. Overlay of 5HT-B reconstructions with different kissing-loop sequences (blue is 5HT-B and red is 5HT-B-V2).
Extended Data Fig. 3 Definition and measurements of angles θ, φ and τ.
a, Schematics showing the definition of crossover angle θ, curvature angle φ and twist angle τ. Diagrams use arrows for 3′ and dots for 5′ when seen from side and circle with a dot for 3′ and circle with a cross for 5′ when seen from end. Direction of angles shown with arrow. Red lines indicate seam base pairs. The seams (S) and helices (H) are numbered from 5′ to 3′. b,c,d, Measurements of θ, φ and τ angles for different RNA origami designs, where angles are identified by seams and helices involved.
Extended Data Fig. 4 Comparison of KL regions and position of adenines.
a, Two views of the instance of bulged-out adenines from our 6HBC-mature dataset. A notable protrusion is present where the adenines are modelled, and a clear lack of density is in the spot where density from base stacking adenines is observed in our highest resolution 5HT-A dataset b, and our lower resolution 5HT-B dataset, c. Adenines shown in red against coulomb potential map shown in cyan. d, A similar gap in density was observed in helix 3 of the 6HB no clasp reconstruction. Left image shows top view. Right image shows side view.
Extended Data Fig. 5 KL motif from X-ray crystallography and cryo-EM.
a, X-ray structure used for modelling RNA origami in silico and b, the model from our cryo-EM data. A line drawn between the phosphate of the first A in the KL motif and 3 nucleotides after the motif is parallel to the helical axis in the EM model, but tilted in the X-ray model. This, along with the P-P distance shows that the KL is compacted and underwound in the cryo-EM structure and can be approximated as 8 bp of A-form helix.
Extended Data Fig. 6 SAXS data and model fitting.
a, SAXS data showing observed scattering pre and post structural transformation of the 6HBC and the predicted scattering from the models prior to rigid-body minimization. b, Fit of the predicted scattering from rigid-body optimized 6HBC-Young and 6HBC-Mature models to the experimental scattering from early and late time points. Black line denotes q = 0.09 Å−1. c, Two views of an overlay of the two conformations (young and mature are turquoise and beige, respectively) of the 6HBC with the cross-strand adenine base stack shown in red.
Extended Data Fig. 7 Cryo-EM images of 16H-Satellite RNA and effect of electron dosage.
a, On a same area of the satellite sample, a series of un-tilted cryo-EM images were acquired under a same illumination condition, for example the electron dose of 50 e−Å−2 for each of six images. Thus, the images are corresponding to radiation damage after 50 e−Å−2, 100 e−Å−2, 150 e−Å−2, 200 e−Å−2, 250 e−Å−2 and 300 e−Å−2, respectively. After a dose above 100 e−Å−2, the radiation damage caused ‘bubbling’ phenomena on the supporting carbon area (left edge). However, the RNA particles in vitrious ice are not showing bubbling even at the dose of 300 e−Å−2. b, Zoomed-in images of three representative areas with particles. The radiation damage blurred the detailed structural features, but the low resolution shape of the particles remains.
Extended Data Fig. 8 IMOD 3D reconstruction from cryo-ET of 16H-satellite.
a, Central slice of the IMOD 3D reconstruction and b, zoomed-in areas that contains 16HS particles. c, The IMOD 3D reconstruction of a selected area shown in the Chimera software and colored based on the height along the normal direction to the plane, where particles are indicated by boxes and d, shows zoomed-in images of selected particles.
Extended Data Fig. 9 IPET 3D reconstruction and effects of masking and low-pass filtering.
IPET cryo-EM 3D reconstruction of two different particles imaged under the dose of 130 e−Å−2 (a) and dose of 68 e−Å−2 (b). Left panels show the central slice of the IPET cryo-EM 3D reconstruction of an individual particle without using the particle-shaped mask. Second left panel shows the central slice using the automatically generated particle-shaped soft mask. Middle panel shows the masked particle low-pass filtered to 60 Å. Second right panel show the final 3D density map. Right panel shows the final 3D density map superimposed with fitted model.
Extended Data Fig. 10 Cryo-ET 3D reconstruction of 16 individual particles of 16H-Satellite RNA.
a, Perpendicular views of the cryo-ET 3D reconstructions with the corresponding fitted models of 8 representative particles. The tilt series was imaged under the total electron dose of 130 e−Å−2. b, Another 8 cryo-ET reconstructions of individual particles that were imaged under the electron dose of 68 e−Å−2. c, Table listing FSC analyses of the final map resolution. Two resolutions were measured for ‘map vs. map’ analysis at FSC = 0.5 and FSC = 0.143 and one resolution was measured for ‘map vs. model’ analysis at FSC = 0.5.
Supplementary Videos 1–4, Tables 1–12 and Figs. 1–31.
Supplementary Video 1
Cryo-EM reconstruction of 5HT-A RNA origami. First, a map from a local refinement using a mask covering the entire structure is shown, autogenerated by cryoSPARC. Colouring has been applied to the map through the motifs modelled into the map. Tetraloops are depicted in yellow, crossovers in blue and KLs in magenta. The map is further refined by local refinement using a mask covering only H2–H4 (shown in grey at the timestamp of 0:47–0:51). This results in better local resolution at the crossovers and central KL. The local resolutions are coloured on the map surface.
Supplementary Video 2
6HBC maturation necessitates the breaking of H6 KL. First, reconstructions of the young and mature 6HBC conformers with the surface near the A2:A2′ stack (coloured in red) are shown. The A2:A2′ stack of the young conformer faces inwards, but the A2:A2′ stack of the mature conformer faces outwards. The rotation of each half of H6 in opposite directions is required to transition between the two conformers; when visualized by interpolation, it becomes clear that the central KL must break for this to occur.
Supplementary Video 3
Local dynamics of 5HT-A RNA origami. Three principal modes of variability were solved using cryoSPARC’s 3D variability analysis algorithms. Each mode is displayed as a volume series comprising 20 different volumes reconstructed from particle sets classified along a given motion trajectory.
Supplementary Video 4
Local dynamics of 6HBC-young and 6HBC-mature conformers. Three principal modes of variability were solved from both 6HBC-young and 6HBC-mature datasets using cryoSPARC’s 3D variability analysis algorithms. Each mode is displayed as a volume series comprising 20 different volumes reconstructed from particle sets classified along a given motion trajectory.
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McRae, E.K.S., Rasmussen, H.Ø., Liu, J. et al. Structure, folding and flexibility of co-transcriptional RNA origami. Nat. Nanotechnol. (2023). https://doi.org/10.1038/s41565-023-01321-6