Letter | Published:

Evolution of a designed protein assembly encapsulating its own RNA genome

Nature volume 552, pages 415420 (21 December 2017) | Download Citation


The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism1 and to display proteins or peptides2,3,4, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a ‘blank slate’ to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies5,6 with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors7,8. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at ‘top-down’ modification of viruses to be safe and effective for drug delivery and vaccine applications1,9,10; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary ‘bottom-up’ approach with considerable advantages in programmability and control.

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We thank R. Chari for RNA-seq advice; S. Bustin for RT–qPCR advice; E. Gray and N. Arroyo for heparinized mouse blood; D. Veesler, J. Kollman and M. Johnson for EM advice; Y. Hsia for DLS advice; C. Walkey, Y. Hsia, G. Rocklin, J. Nelson, A. Chatterjee, S. Kosuri, G. Church, J. Bloom and A. Hessel for suggestions. This work was supported by the Howard Hughes Medical Institute (D.B.), the Bill and Melinda Gates Foundation (D.B. and N.P.K., grant number OPP1118840), the Defense Advanced Research Projects Agency (D.B. and N.P.K., grant number W911NF-15-1-0645), and the NIH (S.H.P., grant number NIH1R01CA177272; D.L.S., grant number NIH1R21NS099654-01A1). G.L.B. was supported by a National Science Foundation Graduate Fellowship. M.J.L. was supported by a Washington Research Foundation Innovation Postdoctoral Fellowship and a Cancer Research Institute Irvington Fellowship from the Cancer Research Institute. H.H.G. was supported by an NIH training grant (NIH5T32HL0071312). U.N. was supported in part by a PHS National Research Service Award (T32GM007270) from NIGMS.

Author information

Author notes

    • Gabriel L. Butterfield
    •  & Marc J. Lajoie

    These authors contributed equally to this work.


  1. Institute for Protein Design, University of Washington, Seattle, Washington 98195, USA

    • Gabriel L. Butterfield
    • , Marc J. Lajoie
    • , Una Nattermann
    • , Daniel Ellis
    • , Jacob B. Bale
    • , Rashmi Ravichandran
    • , Neil P. King
    •  & David Baker
  2. Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA

    • Gabriel L. Butterfield
    • , Marc J. Lajoie
    • , Una Nattermann
    • , Daniel Ellis
    • , Jacob B. Bale
    • , Rashmi Ravichandran
    • , Neil P. King
    •  & David Baker
  3. Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington 98195, USA

    • Gabriel L. Butterfield
    • , Daniel Ellis
    •  & Jacob B. Bale
  4. Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA

    • Heather H. Gustafson
    • , Drew L. Sellers
    • , Sharon Ke
    •  & Suzie H. Pun
  5. Molecular Engineering and Sciences Institute, University of Washington, Seattle, Washington 98195, USA

    • Heather H. Gustafson
    • , Drew L. Sellers
    •  & Suzie H. Pun
  6. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington 98109, USA

    • Drew L. Sellers
  7. Graduate Program in Biological Physics, Structure & Design, University of Washington, Seattle, Washington 98195, USA

    • Una Nattermann
  8. College of Arts & Sciences, University of Washington, Seattle, Washington 98195, USA

    • Garreck H. Lenz
  9. School of Public Health, University of Washington, Seattle, Washington 98195, USA

    • Angelica Yehdego
  10. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA

    • David Baker


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G.L.B. and M.J.L. designed the research and the experimental approach with guidance from N.P.K. and D.B.; G.L.B. and M.J.L. performed the evolution, nucleocapsid characterization, Illumina sequencing, and data analysis; H.H.G. and D.L.S. designed and performed the in vivo mouse experiments, and samples were processed by G.L.B. and M.J.L.; U.N. designed, performed, and analysed electron microscopy experiments; D.E. and J.B.B. designed the starting protein assemblies that were subsequently used for RNA packaging; S.K., G.H.L., A.Y. and R.R. assisted with cloning and protein purification; S.H.P., N.P.K. and D.B. supervised the research; G.L.B. and M.J.L. wrote the manuscript and produced the figures with guidance from H.H.G., D.L.S., U.N., S.H.P., N.P.K. and D.B.; G.L.B., M.J.L., H.H.G., D.L.S., U.N., J.B.B., S.H.P., N.P.K. and D.B. revised the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David Baker.

Reviewer Information Nature thanks D. Schaffer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

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    Life Sciences Reporting Summary

  2. 2.

    Supplementary Information

    This file contains a list of supplemental figures S1-S10, and supplemental tables S1-S6.

Excel files

  1. 1.

    Supplementary Table 1

    This file contains supplementary table 1 - composition of libraries produced for each step of evolution.

  2. 2.

    Supplementary Table 2

    This file contains supplementary table 2 - protein sequences of hydrophilic peptide library for increasing circulation time in live mice.

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