Letter

Biotechnological mass production of DNA origami

Received:
Accepted:
Published online:

Abstract

DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features1,2,3,4,5,6,7,8,9,10,11,12. These structures are customizable in that they can be site-specifically functionalized13 or constructed to exhibit machine-like14,15 or logic-gating behaviour16. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials3,16,17,18,19,20,21,22, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production23; the shorter staple strands are obtained through costly solid-phase synthesis24 or enzymatic processes25. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006)

  2. 2.

    et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009)

  3. 3.

    , , & DNA origami: scaffolds for creating higher order structures. Chem. Rev. 117, 12584–12640 (2017)

  4. 4.

    , & Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009)

  5. 5.

    et al. Designer nanoscale DNA assemblies programmed from the top down. Science 352, 1534 (2016)

  6. 6.

    et al. DNA rendering of polyhedral meshes at the nanoscale. Nature 523, 441–444 (2015)

  7. 7.

    , , & Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components. Science 347, 1446–1452 (2015)

  8. 8.

    & Placing molecules with Bohr radius resolution using DNA origami. Nat. Nanotechnol. 11, 47–52 (2016)

  9. 9.

    , , & Cryo-EM structure of a 3D DNA-origami object. Proc. Natl Acad. Sci. USA 109, 20012–20017 (2012)

  10. 10.

    et al. Complex wireframe DNA origami nanostructures with multi-arm junction vertices. Nat. Nanotechnol. 10, 779–784 (2015)

  11. 11.

    , & Complex shapes self-assembled from single-stranded DNA tiles. Nature 485, 623–626 (2012)

  12. 12.

    , , & Three-dimensional structures self-assembled from DNA bricks. Science 338, 1177–1183 (2012)

  13. 13.

    et al. Routing of individual polymers in designed patterns. Nat. Nanotechnol. 10, 892–898 (2015)

  14. 14.

    , & Nanoscale rotary apparatus formed from tight-fitting 3D DNA components. Sci. Adv. 2, e1501209 (2016)

  15. 15.

    , , & Programmable motion of DNA origami mechanisms. Proc. Natl Acad. Sci. USA 112, 713–718 (2015)

  16. 16.

    , & A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012)

  17. 17.

    , & DNA-nanotube-induced alignment of membrane proteins for NMR structure determination. Proc. Natl Acad. Sci. USA 104, 6644–6648 (2007)

  18. 18.

    et al. Molecular engineering of chiral colloidal liquid crystals using DNA origami. Nat. Mater. 16, 849–856 (2017)

  19. 19.

    et al. Daunorubicin-loaded DNA origami nanostructures circumvent drug-resistance mechanisms in a leukemia model. Small 12, 308–320 (2016)

  20. 20.

    et al. DNA origami delivery system for cancer therapy with tunable release properties. ACS Nano 6, 8684–8691 (2012)

  21. 21.

    et al. DNA origami as a carrier for circumvention of drug resistance. J. Am. Chem. Soc. 134, 13396–13403 (2012)

  22. 22.

    et al. A self-assembled DNA origami-gold nanorod complex for cancer theranostics. Small 11, 5134–5141 (2015)

  23. 23.

    , , & Efficient production of single-stranded phage DNA as scaffolds for DNA origami. Nano Lett. 15, 4672–4676 (2015)

  24. 24.

    Oligo- and poly-nucleotides: 50 years of chemical synthesis. Org. Biomol. Chem. 3, 3851–3868 (2005)

  25. 25.

    , , , & Enzymatic production of ‘monoclonal stoichiometric’ single-stranded DNA oligonucleotides. Nat. Methods 10, 647–652 (2013)

  26. 26.

    , , , & Small, highly active DNAs that hydrolyze DNA. J. Am. Chem. Soc. 135, 9121–9129 (2013)

  27. 27.

    & Production of single-stranded DNAs by self-cleavage of rolling-circle amplification products. Biotechniques 54, 337–343 (2013)

  28. 28.

    et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009)

  29. 29.

    , , & Rapid folding of DNA into nanoscale shapes at constant temperature. Science 338, 1458–1461 (2012)

  30. 30.

    & Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3–11 (1987)

  31. 31.

    et al. A primer to scaffolded DNA origami. Nat. Methods 8, 221–229 (2011)

  32. 32.

    , , & Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures. Nucleic Acids Res. 40, 2862–2868 (2012)

  33. 33.

    , & A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature 427, 618–621 (2004)

  34. 34.

    , & A one pot, one step, precision cloning method with high throughput capability. PLoS One 3, e3647 (2008)

  35. 35.

    , , , & Toward larger DNA origami. Nano Lett. 14, 5740–5747 (2014)

  36. 36.

    , , & Eliminating helper phage from phage display. Nucleic Acids Res. 34, e145 (2006)

  37. 37.

    & Bacteriophages Ch. 8, 77–80 (Springer, 2008)

  38. 38.

    , , , & Specific growth rate and multiplicity of infection affect high-cell-density fermentation with bacteriophage M13 for ssDNA production. Biotechnol. Bioeng. 114, 777–784 (2017)

  39. 39.

    , , & Facile and scalable preparation of pure and dense DNA origami solutions. Angew. Chem. Int. Ed. 53, 12735–12740 (2014)

  40. 40.

    & Molecular Cloning: A Laboratory Manual 3rd edn Vol. 1, Ch. 5 (Cold Spring Harbor, 2001)

  41. 41.

    , , , & Image processing for electron microscopy single-particle analysis using XMIPP. Nat. Protocols 3, 977–990 (2008)

  42. 42.

    et al. Folding complex DNA nanostructures from limited sets of reusable sequences. Nucleic Acids Res. 44, e102 (2016)

Download references

Acknowledgements

We thank D. Maslak (TUM Research Center for Industrial Biotechnology, Technical University of Munich) for cost estimation in pilot-scale production at the TUM Pilot Plant for Industrial Biotechnology. We thank T. Gerling, E. Meier, M. Schickinger and J. Funke for discussions. This project was supported by European Research Council starting grant 256270, the Deutsche Forschungsgemeinschaft through grants provided within TUM IGSSE (Biomat 05 PSN), the Gottfried–Wilhelm–Leibniz Program, the SFB863, and the Excellence Cluster CIPSM (Center for Integrated Protein Science Munich), the ERASynBio project ‘BioOrigami’, funded by Bundesministerium für Bildung und Forschung grant 031 A 458.

Author information

Affiliations

  1. Technical University of Munich, Physics Department and Institute for Advanced Study, Am Coulombwall 4a, 85748 Garching bei München, Germany

    • Florian Praetorius
    • , Benjamin Kick
    • , Maximilian N. Honemann
    •  & Hendrik Dietz
  2. Technical University of Munich, Institute of Biochemical Engineering, Boltzmannstrasse 15, 85748 Garching bei München, Germany

    • Benjamin Kick
    • , Karl L. Behler
    •  & Dirk Weuster-Botz

Authors

  1. Search for Florian Praetorius in:

  2. Search for Benjamin Kick in:

  3. Search for Karl L. Behler in:

  4. Search for Maximilian N. Honemann in:

  5. Search for Dirk Weuster-Botz in:

  6. Search for Hendrik Dietz in:

Contributions

F.P. performed the research. H.D. designed the research. B.K., K.L.B. and D.W.-B. developed and performed the high-cell-density fermentation and large-scale folding procedures. M.N.H. contributed to DNAzyme optimization. F.P. and H.D. wrote the manuscript and prepared the figures. All authors commented on the manuscript.

Competing interests

A provisional patent has been filed that lists F.P. and H.D. as authors.

Corresponding author

Correspondence to Hendrik Dietz.

Reviewer Information Nature thanks M. Famulok and B. Högberg 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

PDF files

  1. 1.

    Supplementary Data 1

    This file contains caDNAno design diagrams of nanorod variants. a Variant with 100 bases per staple shown in Fig. 2a. b Variant with 200 bases per staple shown in Extended Data Fig. 5. c Variant with 400 bases per staple shown in Extended Data Fig. 5.

  2. 2.

    Supplementary Data 2

    This file contains caDNAno design diagrams of screw-nut variants. a Variant with polythymidine passivation shown in Fig. 2b and Fig. 3c. b Variant without polythymidine passivation shown in Fig. 3d.

  3. 3.

    Supplementary Data 3

    caDNAno design diagram of the brick shown in Fig. 2d

  4. 4.

    Supplementary Table 1

    Calculation of cost for material and chemicals of a 1.9-liter fermentation process in a stirred-tank bioreactor.

Excel files

  1. 1.

    Supplementary Table 2

    This file contains sequences of all phagemids used in this work, of the chemically synthesized staples used for the pointer, as well as of all oligonucleotides used in the knock-out experiments.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.