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Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat


Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology—toehold exchange—through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA ‘acrobat’ for rapid movement, we measure a stepping rate constant approaching 1 s−1, which is 10- to 100-fold faster than prior DNA walkers. Finally, we use single-particle tracking to demonstrate movement of the walker over hundreds of nanometres within 10 min, in quantitative agreement with predictions from stepping kinetics. These results suggest that substantial improvements in the operating rates of broad classes of DNA nanomachines utilizing strand displacement are possible.

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Fig. 1: Principle and mechanism of a cartwheeling DNA walker.
Fig. 2: Single-molecule FRET characterization of walkers with varying toehold lengths.
Fig. 3: Characterization of 2D foothold arrays and long-range walker movement.


  1. Sherman, W. B. & Seeman, N. C. A precisely controlled DNA biped walking device. Nano Lett. 4, 1203–1207 (2004).

    Article  CAS  Google Scholar 

  2. Shin, J.-S. & Pierce, N. A. A synthetic DNA walker for molecular transport. J. Am. Chem. Soc. 126, 10834–10835 (2004).

    Article  CAS  Google Scholar 

  3. Tian, Y., He, Y., Chen, Y., Yin, P. & Mao, C. A DNAzyme that walks processively and autonomously along a one-dimensional track. Angew. Chem. Int. Ed. Engl. 44, 4355–4358 (2005).

    Article  CAS  Google Scholar 

  4. Bath, J., Green, S. J. & Turberfield, A. J. A free-running DNA motor powered by a nicking enzyme. Angew. Chem. Int. Ed. Engl. 44, 4358–4361 (2005).

    Article  CAS  Google Scholar 

  5. Omabegho, T., Sha, R. & Seeman, N. C. A bipedal DNA Brownian motor with coordinated legs. Science 324, 67–71 (2009).

    Article  CAS  Google Scholar 

  6. Gu, H., Chao, J., Xiao, S.-J. & Seeman, N. C. A proximity-based programmable DNA nanoscale assembly line. Nature 465, 202–205 (2010).

    Article  CAS  Google Scholar 

  7. He, Y. & Liu, D. R. Autonomous multistep organic synthesis in a single isothermal solution mediated by a DNA walker. Nat. Nanotech. 5, 778–782 (2010).

    Article  CAS  Google Scholar 

  8. Qian, L. & Winfree, E. Scaling up digital circuit computation with DNA strand displacement cascades. Science 332, 1196–1201 (2011).

    Article  CAS  Google Scholar 

  9. Qian, L., Winfree, E. & Bruck, J. Neural network computation with DNA strand displacement cascades. Nature 475, 368–372 (2011).

    Article  CAS  Google Scholar 

  10. Lund, K. et al. Molecular robots guided by prescriptive landscapes. Nature 465, 206–210 (2010).

    Article  CAS  Google Scholar 

  11. Douglas, S. M., Bachelet, I. & Church, G. M. A logic-gated nanorobot for targeted transport of molecular payloads. Science 335, 831–834 (2012).

    Article  CAS  Google Scholar 

  12. Thubagere, A. J. et al. A cargo-sorting DNA robot. Science 357, eaan6558 (2017).

    Article  Google Scholar 

  13. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 3, 103–113 (2011).

    Article  CAS  Google Scholar 

  14. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

    Article  CAS  Google Scholar 

  15. Zhang, D. Y., Chen, S. X. & Yin, P. Optimizing the specificity of nucleic acid hybridization. Nat. Chem. 4, 208–214 (2012).

    Article  CAS  Google Scholar 

  16. Fu, J. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotech. 9, 531–536 (2014).

    Article  CAS  Google Scholar 

  17. Wickham, S. F. J. et al. Direct observation of stepwise movement of a synthetic molecular transporter. Nat. Nanotech. 6, 166–169 (2011).

    Article  CAS  Google Scholar 

  18. Cha, T.-G. et al. A synthetic DNA motor that transports nanoparticles along carbon nanotubes. Nat. Nanotech. 9, 39–43 (2014).

    Article  CAS  Google Scholar 

  19. Yehl, K. et al. High-speed DNA-based rolling motors powered by RNase H. Nat. Nanotech. 11, 184–190 (2015).

    Article  Google Scholar 

  20. King, S. J. & Schroer, T. A. Dynactin increases the processivity of the cytoplasmic dynein motor. Nat. Cell Biol. 2, 20–24 (2000).

    Article  CAS  Google Scholar 

  21. Kural, C. et al. Kinesin and dynein move a peroxisome in vivo: a tug-of-war or coordinated movement? Science 308, 1469–1472 (2005).

    Article  CAS  Google Scholar 

  22. Pan, J., Li, F., Cha, T.-G., Chen, H. & Choi, J. H. Recent progress on DNA based walkers. Curr. Opin. Biotechnol. 34, 56–64 (2015).

    Article  Google Scholar 

  23. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  24. Walter, N. G., Huang, C.-Y., Manzo, A. J. & Sobhy, M. A. Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat. Methods 5, 475–489 (2008).

    Article  CAS  Google Scholar 

  25. Srinivas, N. et al. On the biophysics and kinetics of toehold-mediated DNA strand displacement. Nucleic Acids Res. 41, 10641–10658 (2013).

    Article  CAS  Google Scholar 

  26. Panyutin, I. G. & Hsieh, P. Formation of a single base mismatch impedes spontaneous DNA branch migration. J. Mol. Biol. 230, 413–424 (1993).

    Article  CAS  Google Scholar 

  27. Beattie, K. L., Wiegand, R. C. & Radding, C. M. Uptake of homologous single-stranded fragments by superhelical DNA. J. Mol. Biol. 116, 783–803 (1977).

    Article  CAS  Google Scholar 

  28. Jungmann, R. et al. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756–4761 (2010).

    Article  CAS  Google Scholar 

  29. Dupuis, N. F., Holmstrom, E. D. & Nesbitt, D. J. Single-molecule kinetics reveal cation-promoted DNA duplex formation through ordering of single-stranded helices. Biophys. J. 105, 756–766 (2013).

    Article  CAS  Google Scholar 

  30. Smith, S. B., Cui, Y. & Bustamante, C. Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271, 795–799 (1996).

    Article  CAS  Google Scholar 

  31. Helenius, J., Brouhard, G., Kalaidzidis, Y., Diez, S. & Howard, J. The depolymerizing kinesin MCAK uses lattice diffusion to rapidly target microtubule ends. Nature 441, 115–119 (2006).

    Article  CAS  Google Scholar 

  32. Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001).

    Article  CAS  Google Scholar 

  33. Michelotti, N., de Silva, C., Johnson-Buck, A. E., Manzo, A. J. & Walter, N. G. A bird’s eye view tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods Enzymol. 475, 121–148 (2010).

    Article  CAS  Google Scholar 

  34. Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008).

    Article  CAS  Google Scholar 

  35. Blanco, M. & Walter, N. G. Analysis of complex single-molecule FRET time trajectories. Methods Enzymol. 472, 153–178 (2010).

    Article  CAS  Google Scholar 

  36. Nicolai, C. & Sachs, F. Solving ion channel kinetics with the qub software. Biophys. Rev. Lett. 08, 191–211 (2013).

    Article  Google Scholar 

  37. Dill, K. A. & Bromberg, S. Molecular Driving Forces: Statistical Thermodynamics in Chemistry and Biology. (Garland Science: New York, 2003).

    Google Scholar 

  38. Chen, H. et al. Ionic strength-dependent persistence lengths of single-stranded RNA and DNA. Proc. Natl Acad. Sci. USA 109, 799–804 (2012).

    Article  CAS  Google Scholar 

  39. Johnson-Buck, A. et al. Kinetic fingerprinting to identify and count single nucleic acids. Nat. Biotechnol. 33, 730–732 (2015).

    Article  CAS  Google Scholar 

  40. Particle Track and Analysis (PTA) (Yoshiyuki Arai, accessed 29 August 2017);

  41. Gillespie, D. T. Exact stochastic simulation of coupled chemical reactions. J. Phys. Chem. 81, 2340–2361 (1977).

    Article  CAS  Google Scholar 

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This work was supported primarily by the US Department of Defense Army Research Office MURI award W911NF-12-1-0420 to N.G.W. and H.Y. The authors thank J. Damon Hoff for technical support, Z. R. Li for graphic design support, and B. Nijholt, E. Krieg, A. M. Bergman and W. Benjamin Rogers for discussions about DNA origami design.

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Authors and Affiliations



A.J.-B. conceived the ideas. Y.R.Y. designed, fabricated and characterized the DNA tile samples. W.M.S. and A.J.-B. designed, fabricated and characterized the DNA origami samples. J.L. and A.J.-B. performed smFRET and single-particle tracking measurements. J.L., A.J.-B., and N.G.W. analysed and interpreted the data. J.L. and A.J.-B. co-wrote the paper, and all authors discussed the results and edited the manuscript.

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Correspondence to Nils G. Walter.

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Supplementary Text, Supplementary Figures 1–34, Supplementary Tables 1–5 and Supplementary References

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Li, J., Johnson-Buck, A., Yang, Y.R. et al. Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat. Nature Nanotech 13, 723–729 (2018).

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