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Computational design of three-dimensional RNA structure and function

Nature Nanotechnology volume 14, pages 866–873 (2019)Cite this article

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Abstract

RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.

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Fig. 1: Problems in RNA nanotechnology reduced to RNA motif pathfinding problems and solved by RNAMake.
Fig. 2: Solving the TTR design problem.
Fig. 3: The ribosome tethering problem.
Fig. 4: Stabilizing aptamers for ATP and light-up fluorophores through designer tertiary contacts.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request. Furthermore, all of our chemical mapping data are available on https://rmdb.stanford.edu, and a detailed table of the accession identifications is given in the Supplementary Information.

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Acknowledgements

We thank S. Bonilla for assistance in performing the native gel assays and A. Watkins for discussions about ribosome tether design. We thank the Straight lab for graciously providing X. laevis whole cell lysate. This work was supported by the National Institutes of Health, through NIGMS MIRA R35 GM122579 (R.D.), R01 GM121487 (R.D.), New Innovator Award 1DP2GM110838 (J.B.L.), Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowships GM112294 (J.D.Y.) and GM100953 (D.E.), P01 GM066275 (D.H.) and R35 GM118070 (J.S.K.), a Stanford School of Medicine Discovery Innovation Award (R.D.), Army Research Office W911NF-16-1-0372 (M.C.J.), the National Science Foundation through MCB-1716766 (M.C.J.), Career Award 1452441 (J.B.L.) and Graduate Research Fellowship DGE-1324585 (A.E.D.), the David and Lucile Packard Foundation (M.C.J.) and the Camille Dreyfus Teacher-Scholar Program (M.C.J.).

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA

    Joseph D. Yesselman, Michael R. Gotrik, Wipapat Kladwang, Xuesong Shi, Daniel Herschlag & Rhiju Das

  2. Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, CO, USA

    Daniel Eiler, David A. Costantino & Jeffrey S. Kieft

  3. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Erik D. Carlson, Anne E. d’Aquino, Julius B. Lucks & Michael C. Jewett

  4. Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, USA

    Erik D. Carlson, Julius B. Lucks & Michael C. Jewett

  5. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Erik D. Carlson, Anne E. d’Aquino, Julius B. Lucks & Michael C. Jewett

  6. Interdisciplinary Biological Sciences Graduate Program, Northwestern University, Evanston, IL, USA

    Anne E. d’Aquino, Julius B. Lucks & Michael C. Jewett

  7. Department of Cancer Genetics & Genomics, Stanford University School of Medicine, Stanford, CA, USA

    Alexandra N. Ooms

  8. Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA

    Paul D. Carlson

  9. Department of Chemistry, Stanford University School of Medicine, Stanford, CA, USA

    Daniel Herschlag

  10. Stanford ChEM-H (Chemistry, Engineering, and Medicine for Human Health), Stanford University, Stanford, CA, USA

    Daniel Herschlag

  11. Department of Physics, Stanford University, Stanford, CA, USA

    Rhiju Das

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  1. Joseph D. Yesselman
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Contributions

R.D. and J.D.Y. conceived the study. J.D.Y. developed RNAMake and generated the models and sequences used throughout the study. A.N.O., W.K. and J.D.Y. performed the chemical mapping, titrations and native gel assays for miniTTR constructs. X.S. and D.H. designed and performed the SAXS on miniTTR 2 and 6. D.E. solved the miniTTR crystal structure assisted by D.A.C. and J.S.K. in preparing the RNA and analysis. E.D.C., A.E.D. and M.C.J. made and tested the RNAMake-designed ribosomes P.D.C. and J.B.L. carried out the SHAPE-seq and in vivo tests of Spinach-TTRs. M.R.G. performed fluorescence and lysate experiments on Spinach-TTRs. J.D.Y. and R.D. wrote the paper, with participation by all the authors.

Corresponding author

Correspondence to Rhiju Das.

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Competing interests

Stanford University is filing a patent on aptamer stabilization with RNAMake.

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Peer review information: Nature Nanotechnology thanks Peixuan Guo, Nils (G) Walter and other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Results, Supplementary Discussion, Supplementary Methods, Supplementary Tables 1–10, Supplementary Figs. 1–14 and Supplementary refs. 1–46.

All sequences used in this study.

All primers used in this study.

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Yesselman, J.D., Eiler, D., Carlson, E.D. et al. Computational design of three-dimensional RNA structure and function. Nat. Nanotechnol. 14, 866–873 (2019). https://doi.org/10.1038/s41565-019-0517-8

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