Programmable autonomous synthesis of single-stranded DNA

Abstract

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: PER overview.
Figure 2: PER mechanism.
Figure 3: PER cascades.
Figure 4: PER nanodevice for conditional RNA degradation.
Figure 5: Signal amplifier with PER.
Figure 6: Logic computation with PER.
Figure 7: PER temporal molecular event recorder.

References

  1. 1

    Winfree, E., Liu, F., Wenzler, L. A. & Seeman, N. C. Design and self-assembly of two-dimensional DNA crystals. Nature 394, 539–544 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

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

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

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

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Zheng, J. et al. From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal. Nature 461, 74–77 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Wei, B., Dai, M. & Yin, P. Complex shapes self-assembled from single-stranded DNA tiles. Nature 485, 623–626 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Ke, Y., Ong, L. L., Shih, W. M. & Yin, P. Three-dimensional structures self-assembled from DNA bricks. Science 338, 1177–1183 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Han, D. et al. DNA gridiron nanostructures based on four-arm junctions. Science 339, 1412–1415 (2013).

    CAS  PubMed  Google Scholar 

  8. 8

    Gerling, T., Wagenbauer, K. F., Neuner, A. M. & Dietz, H. Dynamic DNA devices and assemblies formed by shape-complementary, non-base pairing 3D components. Science 347, 1446–1452 (2015).

    CAS  PubMed  Google Scholar 

  9. 9

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Dunn, K. E. et al. Guiding the folding pathway of DNA origami. Nature 525, 82–86 (2015).

    CAS  PubMed  Google Scholar 

  11. 11

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

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1588 (2006).

    CAS  PubMed  Google Scholar 

  13. 13

    Zhang, D. Y., Turberfield, A. J., Yurke, B. & Winfree, E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science 318, 1121–1125 (2007).

    CAS  PubMed  Google Scholar 

  14. 14

    Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).

    CAS  PubMed  Google Scholar 

  15. 15

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

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

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

    CAS  PubMed  Google Scholar 

  17. 17

    Chirieleison, S. M., Allen, P. B., Simpson, Z. B., Ellington, A. D. & Chen, X. Pattern transformation with DNA circuits. Nat. Chem. 5, 1000–1005 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Weitz, M. et al. Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator. Nat. Chem. 6, 295–302 (2014).

    CAS  PubMed  Google Scholar 

  19. 19

    Karzbrun, E., Tayar, A. M., Noireaux, V. & Bar-Ziv, R. H. Programmable on-chip DNA compartments as artificial cells. Science 345, 829–832 (2014).

    CAS  PubMed  Google Scholar 

  20. 20

    Mohammed, A. M., Šulc, P., Zenk, J. & Schulman, R. Self-assembling DNA nanotubes to connect molecular landmarks. Nat. Nanotech. 12, 312–316 (2016).

    Google Scholar 

  21. 21

    Choi, H. M. T. et al. Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat. Biotechnol. 28, 1208–1212 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

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

    CAS  PubMed  Google Scholar 

  24. 24

    Kuzyk, A. et al. DNA-based self-assembly of chiral plasmonic nanostructures with tailored optical response. Nature 483, 311–314 (2012).

    CAS  PubMed  Google Scholar 

  25. 25

    Derr, N. D. et al. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338, 662–665 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Sun, W. et al. Casting inorganic structures with DNA molds. Science 346, 1258361 (2014).

    PubMed  PubMed Central  Google Scholar 

  28. 28

    Wang, J. S. & Zhang, D. Y. Simulation-guided DNA probe design for consistently ultraspecific hybridization. Nat. Chem. 7, 545–553 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Gopinath, A., Miyazono, E., Faraon, A. & Rothemund, P. W. K. Engineering and mapping nanocavity emission via precision placement of DNA origami. Nature 535, 401–405 (2016).

    CAS  PubMed  Google Scholar 

  30. 30

    Bhatia, D. et al. Quantum dot-loaded monofunctionalized DNA icosahedra for single-particle tracking of endocytic pathways. Nat. Nanotech. 11, 1112–1119 (2016).

    CAS  Google Scholar 

  31. 31

    Kilchherr, F. et al. Single-molecule dissection of stacking forces in DNA. Science 353, aaf5508 (2016).

    PubMed  Google Scholar 

  32. 32

    Nickels, P. C. et al. Molecular force spectroscopy with a DNA origami-based nanoscopic force clamp. Science 354, 305–307 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Walker, G. T. et al. Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 20, 1691–1696 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Lizardi, P. M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225–232 (1998).

    CAS  PubMed  Google Scholar 

  35. 35

    Notomi, T. et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Du, Y. & Dong, S. Nucleic acid biosensors: recent advances and perspectives. Anal. Chem. 89, 189–215 (2017).

    CAS  PubMed  Google Scholar 

  38. 38

    Church, G. M., Gao, Y. & Kosuri, S. Next-generation digital information storage in DNA. Science 337, 1628 (2012).

    CAS  PubMed  Google Scholar 

  39. 39

    Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    CAS  PubMed  Google Scholar 

  40. 40

    Saiki, R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491 (1988).

    CAS  PubMed  Google Scholar 

  41. 41

    Montagne, K., Plasson, R., Sakai, Y., Fujii, T. & Rondelez, Y. Programming an in vitro DNA oscillator using a molecular networking strategy. Mol. Syst. Biol. 7, 466 (2011).

    PubMed  PubMed Central  Google Scholar 

  42. 42

    Baccouche, A., Montagne, K., Padirac, A., Fujii, T. & Rondelez, Y. Dynamic DNA-toolbox reaction circuits: a walkthrough. Methods 67, 234–249 (2014).

    CAS  PubMed  Google Scholar 

  43. 43

    Lee, C. S., Davis, R. W. & Davidson, N. A physical study by electron microscopy of the terminally repetitious, circularly permuted DNA from the coliphage particles of Escherichia coli 15. J. Mol. Biol. 48, 1–22 (1970).

    CAS  PubMed  Google Scholar 

  44. 44

    Schaus, T. E., Woo, S., Xuan, F., Chen, X. & Yin, P. A DNA nanoscope via auto-cycling proximity recording. Nat. Commun. 8, 696 (2017).

    PubMed  PubMed Central  Google Scholar 

  45. 45

    Newton, C. R. et al. The production of PCR products with 5′ single-stranded tails using primers that incorporate novel phosphoramidite intermediates. Nucleic Acids Res. 21, 1155–1162 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Sakamoto, K. et al. State transitions by molecules. Biosystems 52, 81–91 (1999).

    CAS  PubMed  Google Scholar 

  47. 47

    Whitcombe, D., Theaker, J., Guy, S. P., Brown, T. & Little, S. Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17, 804–807 (1999).

    CAS  PubMed  Google Scholar 

  48. 48

    Aubert, N., Rondelez, Y., Fujii, T. & Hagiya, M. Enforcing logical delays in DNA computing systems. Nat. Comput. 13, 559–572 (2014).

    CAS  Google Scholar 

  49. 49

    Grillari, J., Hackl, M. & Grillari-Voglauer, R. miR-17-92 cluster: ups and downs in cancer and aging. Biogerontology 11, 501–506 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Hjiantoniou, E., Iseki, S., Uney, J. B. & Phylactou, L. A. DNAzyme-mediated cleavage of twist transcripts and increase in cellular apoptosis. Biochem. Biophys. Res. Commun. 300, 178–181 (2003).

    CAS  PubMed  Google Scholar 

  51. 51

    Santoro, S. W. & Joyce, G. F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl Acad. Sci. USA 94, 4262–4266 (1997).

    CAS  PubMed  Google Scholar 

  52. 52

    Mohanty, J. et al. Thioflavin T as an efficient inducer and selective fluorescent sensor for the human telomeric G-quadruplex DNA. J. Am. Chem. Soc. 135, 367–376 (2012).

    PubMed  Google Scholar 

  53. 53

    Yurke, B., Turberfield, A. J., Mills, A. P., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605–608 (2000).

    CAS  PubMed  Google Scholar 

  54. 54

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

    CAS  PubMed  Google Scholar 

  55. 55

    Hagiya, M., Arita, M., Kiga, D., Sakamoto, K. & Yokoyama, S. in DNA Based Computers III Vol. 48 (eds Rubin, H. & Wood, D. H.) 57–72 (DIMACS Series in Discrete Mathematics and Theoretical Computer Science, American Mathematical Society, 1999).

    Google Scholar 

  56. 56

    Winfree, E. Whiplash PCR for O(1) Computing Technical Report 1998.23 (Caltech, 1998).

  57. 57

    Rose, J. A., Deaton, R. J., Hagiya, M. & Suyama, A. in DNA Computing (Jonoska, N. & Seeman, N. C.) 104–116 (Springer, 2002).

    Google Scholar 

  58. 58

    Komiya, K., Yamamura, M. & Rose, J. A. in International Workshop on DNA-Based Computers 1–10 (Springer, 2008).

    Google Scholar 

  59. 59

    Reif, J. H. & Majumder, U. Isothermal reactivating whiplash PCR for locally programmable molecular computation. Nat. Comput. 9, 183–206 (2010).

    CAS  Google Scholar 

  60. 60

    Fujii, T. & Rondelez, Y. Predator–prey molecular ecosystems. ACS Nano 7, 27–34 (2012).

    PubMed  Google Scholar 

  61. 61

    Padirac, A., Fujii, T., Estévez-Torres, A. & Rondelez, Y. Spatial waves in synthetic biochemical networks. J. Am. Chem. Soc. 135, 14586–14592 (2013).

    CAS  PubMed  Google Scholar 

  62. 62

    Zadorin, A. S., Rondelez, Y., Galas, J.-C. & Estevez-Torres, A. Synthesis of programmable reaction-diffusion fronts using DNA catalyzers. Phys. Rev. Lett. 114, 068301 (2015).

    CAS  PubMed  Google Scholar 

  63. 63

    Dirks, R. M. & Pierce, N. A. Triggered amplification by hybridization chain reaction. Proc. Natl Acad. Sci. USA 101, 15275–15278 (2004).

    CAS  PubMed  Google Scholar 

  64. 64

    Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Dirks, R. M., Bois, J. S., Schaeffer, J. M., Winfree, E. & Pierce, N. A. Thermodynamic analysis of interacting nucleic acid strands. SIAM Rev. 49, 65–88 (2007).

    Google Scholar 

  66. 66

    Zadeh, J. N. et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).

    CAS  PubMed  Google Scholar 

  67. 67

    Wolfe, B. R. & Pierce, N. A. Sequence design for a test tube of interacting nucleic acid strands. ACS Synth. Biol. 4, 1086–1100 (2014).

    PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank W. Shih, J. Kim, X. Chen, N. Hanikel, E. Winfree, B. Beliveau and N. Liu for their discussions and comments. This work was supported by the Office on Naval Research (grants N000141310593, N000141410610, N000141612182 and N000141612410), the National Science Foundation (grants CCF1317291, CMMI1334109 and 1540214), the National Institutes of Health (grant 1R01EB01865901) and the Wyss Institute's Molecular Robotics Initiative. J. Kishi was supported by an NSF graduate research fellowship and T. Schaus was supported by the Jane Coffin Childs Postdoctoral Fellowship.

Author information

Affiliations

Authors

Contributions

J.Y.K. conceived and designed the study, designed and performed the experiments, analysed the data and wrote the manuscript. T.E.S. designed and performed the experiments and analysed the data. N.G. designed and performed the experiments and analysed the data. F.X. designed and performed the experiments and analysed the data. P.Y. conceived and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed, edited and approved the manuscript.

Corresponding author

Correspondence to Peng Yin.

Ethics declarations

Competing interests

A provisional US patent has been filed based on this work. P.Y. is co-founder of Ultivue Inc. and NuProbe Global.

Supplementary information

Supplementary information

Supplementary information (PDF 21412 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kishi, J., Schaus, T., Gopalkrishnan, N. et al. Programmable autonomous synthesis of single-stranded DNA. Nature Chem 10, 155–164 (2018). https://doi.org/10.1038/nchem.2872

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing