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Published June 2021 | Accepted Version + Supplemental Material
Journal Article Open

RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds


RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.

Additional Information

© 2021 Nature Publishing Group. Received 19 October 2019; Accepted 08 March 2021; Published 10 May 2021. We thank L. Qian and E. Winfree for use of their atomic force microscopes, G. Tikhomirov for help with AFM and M. Jepsen for help with FRET. We acknowledge the EteRNA community for conducting an experiment that suggested that kissing loop sequences are less constrained than previously assumed. This inspired us to add de novo design of KLs to Revolvr. C.G. acknowledges a fellowship from the Carlsberg Research Foundation. E.K.S.M. acknowledges the Natural Sciences and Engineering Research Council of Canada for his post doctoral fellowship. P.W.K.R. acknowledges funding by NSF grants (CCF-1317694 and CMMI-1636364) and ONR grants (N00014-16-1-2159, N00014-17-1-2610 and N00014-18-1-2649). E.S.A. acknowledges funding by the ERC Consolidator Grant (RNA ORIGAMI—RNA-protein nanostructures for synthetic biology, 683305), which supported the work of C.G., G.G. and E.K.S.M., and the Independent Research Fund Denmark (9040-00425B), which supported the work of E.K.S.M. Data availability: The data supporting the findings of this study are further documented in the associated Supplementary Information. All raw data and analysis files used in the study are available upon request from the authors. Code availability: The code used to generate RNA origami designs in this study is included in the associated Supplementary Information. Future updates to the code will be made available on GitHub (https://github.com/esa-lab/ROAD) and on a dedicated web server with accompanying tutorials (https://bion.au.dk/software/rnao-design/). The code is licensed under the MIT licence. Author Contributions: C.G., P.W.K.R. and E.S.A. conceived the project. C.G., G.G. and E.K.S.M. performed the research. P.W.K.R. and E.S.A. supervised the project. C.G., P.W.K.R. and E.S.A. wrote the manuscript. All authors analysed the data and commented on the manuscript. The authors declare no competing interests. Peer review information: Nature Chemistry thanks Kirill Afonin, Hendrik Dietz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Attached Files

Accepted Version - EMS118922.pdf

Supplemental Material - 41557_2021_679_MOESM1_ESM.pdf

Supplemental Material - 41557_2021_679_MOESM2_ESM.zip

Supplemental Material - 41557_2021_679_MOESM3_ESM.mov

Supplemental Material - 41557_2021_679_MOESM4_ESM.mov

Supplemental Material - 41557_2021_679_MOESM5_ESM.mov

Supplemental Material - 41557_2021_679_MOESM6_ESM.mov

Supplemental Material - 41557_2021_679_MOESM7_ESM.mov

Supplemental Material - 41557_2021_679_MOESM8_ESM.mov


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Additional details

August 22, 2023
October 23, 2023