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

Nature Chemistry volume 13, pages 549–558 (2021)Cite this article

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Abstract

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.

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Fig. 1: Modular design of RNA origami.
Fig. 2: Design of RNA origami curvature and tile–tile interfaces.
Fig. 3: Building taller and wider RNA origami scaffolds.
Fig. 4: TEM analysis of RNA origami structures with optimal and suboptimal designs.
Fig. 5: Scaffolding fluorescent proteins and small molecules on RNA origami.

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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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Interdisciplinary Nanoscience Center and Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Cody Geary, Guido Grossi, Ewan K. S. McRae & Ebbe S. Andersen

  2. Bioengineering, Computing + Mathematical Sciences, and Computation & Neural Systems, California Institute of Technology, Pasadena, CA, USA

    Cody Geary & Paul W. K. Rothemund

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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.

Corresponding authors

Correspondence to Paul W. K. Rothemund or Ebbe S. Andersen.

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The authors declare no competing interests.

Additional information

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.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–31, Supplementary Tables 1–9 and Supplementary Note 1.

Supplementary Software

ROAD software package containing Perl scripts, example data, documentation and tutorial.

Supplementary Video 1

Animation of cotranscriptional folding of ZigZag-B-4X made using RNApath and Chimera.

Supplementary Video 2

Animation of cotranscriptional folding of Ribbon-9H made using RNApath and Chimera.

Supplementary Video 3

Animation of cotranscriptional folding of Path1 made using RNApath and Chimera.

Supplementary Video 4

Animation of cotranscriptional folding of Path2 made using RNApath and Chimera.

Supplementary Video 5

Animation of cotranscriptional folding of Barriers_Example1 made using RNApath and Chimera.

Supplementary Video 6

Animation of cotranscriptional folding of Barriers_Example2 made using RNApath and Chimera.

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Geary, C., Grossi, G., McRae, E.K.S. et al. RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds. Nat. Chem. 13, 549–558 (2021). https://doi.org/10.1038/s41557-021-00679-1

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