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Rolling circle amplification (RCA)-based DNA hydrogel


DNA hydrogels have unique properties, including sequence programmability, precise molecular recognition, stimuli-responsiveness, biocompatibility and biodegradability, that have enabled their use in diverse applications ranging from material science to biomedicine. Here, we describe a rolling circle amplification (RCA)-based synthesis of 3D DNA hydrogels with rationally programmed sequences and tunable physical, chemical and biological properties. RCA is a simple and highly efficient isothermal enzymatic amplification strategy to synthesize ultralong single-stranded DNA that benefits from mild reaction conditions, and stability and efficiency in complex biological environments. Other available methods for synthesis of DNA hydrogels include hybridization chain reactions, which need a large amount of hairpin strands to produce DNA chains, and PCR, which requires temperature cycling. In contrast, the RCA process is conducted at a constant temperature and requires a small amount of circular DNA template. In this protocol, the polymerase phi29 catalyzes the elongation and displacement of DNA chains to amplify DNA, which subsequently forms a 3D hydrogel network via various cross-linking strategies, including entanglement of DNA chains, multi-primed chain amplification, hybridization between DNA chains, and hybridization with functional moieties. We also describe how to use the protocol for isolation of bone marrow mesenchymal stem cells and cell delivery. The whole protocol takes ~2 d to complete, including hydrogel synthesis and applications in cell isolation and cell delivery.

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Fig. 1: Overview of the protocol for the preparation of RCA-based DNA hydrogels.
Fig. 2: RCA-based DNA network for stem cell isolation from bone marrow.
Fig. 3: RCA-based DNA robot for cell delivery in confined space.
Fig. 4: Synthesis of DNA hydrogel by entanglement of DNA chains.
Fig. 5: Synthesis of DNA hydrogel by multi-primed chain amplification.
Fig. 6: Synthesis of DNA hydrogel by hybridization between DNA chains.
Fig. 7: Synthesis of DNA hydrogel by hybridization with functional moieties.
Fig. 8: Characterization of RCA-based hydrogels.

Data availability

The main data supporting the examples of this protocol are available in the supporting primary research papers22,23,24. Additional data are available from the corresponding author upon reasonable request.


  1. Bao, M., Xie, J. & Huck, W. T. S. Recent advances in engineering the stem cell microniche in 3D. Adv. Sci. 5, 1800448 (2018).

    Article  CAS  Google Scholar 

  2. Chaudhuri, O. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326–334 (2016).

    Article  CAS  Google Scholar 

  3. Kraehenbuehl, T. P., Langer, R. & Ferreira, L. S. Three-dimensional biomaterials for the study of human pluripotent stem cells. Nat. Methods 8, 731–736 (2011).

    Article  CAS  Google Scholar 

  4. Baker, B. M. et al. Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments. Nat. Mater. 14, 1262–1268 (2015).

    Article  CAS  Google Scholar 

  5. Zhang, Y. S. & Khademhosseini, A. Advances in engineering hydrogels. Science 356, eaaf3627 (2017).

    Article  CAS  Google Scholar 

  6. Yuk, H., Lu, B. & Zhao, X. Hydrogel bioelectronics. Chem. Soc. Rev. 48, 1642–1667 (2019).

    Article  CAS  Google Scholar 

  7. Culver, H. R., Clegg, J. R. & Peppas, N. A. Analyte-responsive hydrogels: Intelligent materials for biosensing and drug delivery. Acc. Chem. Res. 50, 170–178 (2017).

    Article  CAS  Google Scholar 

  8. Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

    Article  CAS  Google Scholar 

  9. Yang, D. et al. DNA materials: bridging nanotechnology and biotechnology. Acc. Chem. Res. 47, 1902–1911 (2014).

    Article  CAS  Google Scholar 

  10. Roh, Y. H., Ruiz, R. C., Peng, S., Lee, J. B. & Luo, D. Engineering DNA-based functional materials. Chem. Soc. Rev. 40, 5730–5744 (2011).

    Article  CAS  Google Scholar 

  11. Wang, D., Hu, Y., Liu, P. & Luo, D. Bioresponsive DNA hydrogels: beyond the conventional stimuli responsiveness. Acc. Chem. Res. 50, 733–739 (2017).

    Article  CAS  Google Scholar 

  12. Shao, Y., Jia, H., Cao, T. & Liu, D. Supramolecular hydrogels based on DNA self-assembly. Acc. Chem. Res. 50, 659–668 (2017).

    Article  CAS  Google Scholar 

  13. Kahn, J. S., Hu, Y. & Willner, I. Stimuli-responsive DNA-based hydrogels: from basic principles to applications. Acc. Chem. Res. 50, 680–690 (2017).

    Article  CAS  Google Scholar 

  14. Li, J. et al. Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy. J. Am. Chem. Soc. 137, 1412–1415 (2015).

    Article  CAS  Google Scholar 

  15. Wang, J. et al. Clamped hybridization chain reactions for the self-assembly of patterned DNA hydrogels. Angew. Chem. Int. Ed. 56, 2171–2175 (2017).

    Article  CAS  Google Scholar 

  16. Nöll, T. et al. Construction of three-dimensional DNA hydrogels from linear building blocks. Angew. Chem. Int. Ed. 53, 8328–8332 (2014).

    Article  CAS  Google Scholar 

  17. Li, J. et al. Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications. Chem. Soc. Rev. 45, 1410–1431 (2016).

    Article  CAS  Google Scholar 

  18. English, M. A. et al. Programmable crispr-responsive smart materials. Science 365, 780–785 (2019).

    Article  CAS  Google Scholar 

  19. Li, F., Tang, J., Geng, J., Luo, D. & Yang, D. Polymeric DNA hydrogel: Design, synthesis and applications. Prog. Polym. Sci. 98, 101163 (2019).

    Article  CAS  Google Scholar 

  20. Um, S. H. et al. Enzyme-catalysed assembly of DNA hydrogel. Nat. Mater. 5, 797–801 (2006).

    Article  CAS  Google Scholar 

  21. Cheng, E. et al. A pH-triggered, fast-responding DNA hydrogel. Angew. Chem. Int. Ed. 48, 7660–7663 (2009).

    Article  CAS  Google Scholar 

  22. Lee, J. B. et al. A mechanical metamaterial made from a DNA hydrogel. Nat. Nanotechnol. 7, 816–820 (2012).

    Article  CAS  Google Scholar 

  23. Yao, C. et al. Double rolling circle amplification generates physically cross-linked DNA network for stem cell fishing. J. Am. Chem. Soc. 142, 3422–3429 (2020).

    Article  CAS  Google Scholar 

  24. Tang, J. et al. Super-soft and super-elastic DNA robot with magnetically-driven navigational locomotion for cell delivery in confined space. Angew. Chem. Int. Ed. 59, 2490–2495 (2020).

    Article  CAS  Google Scholar 

  25. Geng, J. et al. A fluorescent biofunctional DNA hydrogel prepared by enzymatic polymerization. Adv. Healthc. Mater. 7, 1700998 (2018).

    Article  CAS  Google Scholar 

  26. Merindol, R., Delechiave, G., Heinen, L., Catalani, L. H. & Walther, A. Modular design of programmable mechanofluorescent DNA hydrogels. Nat. Commun. 10, 528 (2019).

    Article  CAS  Google Scholar 

  27. Hamada, S. et al. Dynamic DNA material with emergent locomotion behavior powered by artificial metabolism. Sci. Robot. 4, eaaw3512 (2019).

    Article  Google Scholar 

  28. Madl, C. M., Heilshorn, S. C. & Blau, H. M. Bioengineering strategies to accelerate stem cell therapeutics. Nature 557, 335–342 (2018).

    Article  CAS  Google Scholar 

  29. De Luca, M. et al. Advances in stem cell research and therapeutic development. Nat. Cell Biol. 21, 801–811 (2019).

    Article  CAS  Google Scholar 

  30. Qian, X. et al. Untethered recyclable tubular actuators with versatile locomotion for soft continuum robots. Adv. Mater., e1801103 (2018).

  31. Wang, C. et al. Soft ultrathin electronics innervated adaptive fully soft robots. Adv. Mater. 30, e1706695 (2018).

    Article  CAS  Google Scholar 

  32. Cangialosi, A. et al. DNA sequence-directed shape change of photopatterned hydrogels via high-degree swelling. Science 357, 1126–1130 (2017).

    Article  CAS  Google Scholar 

  33. Yang, C. & Suo, Z. Hydrogel ionotronics. Nat. Rev. Mater. 3, 125–142 (2018).

    Article  CAS  Google Scholar 

  34. Gao, T. et al. Design and fabrication of flexible DNA polymer cocoons to encapsulate live cells. Nat. Commun. 10, 2946 (2019).

    Article  CAS  Google Scholar 

  35. Ye, D. et al. Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction. Nat. Protoc. 15, 2163–2185 (2020).

    Article  CAS  Google Scholar 

  36. Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  Google Scholar 

  37. Li, L. et al. Nucleic acid aptamers for molecular diagnostics and therapeutics: Advances and perspectives. Angew. Chem. Int. Ed. Engl. 60, 2221–2231 (2021).

    Article  CAS  Google Scholar 

  38. McCarthy, S. A., Davies, G. L. & Gun’ko, Y. K. Preparation of multifunctional nanoparticles and their assemblies. Nat. Protoc. 7, 1677–1693 (2012).

    Article  CAS  Google Scholar 

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This work was supported in part by National Natural Science Foundation of China (21621004 and 21704074), National Key R&D Program of China (2019YFA09005800 and 2018YFA0902300) and the Tianjin Natural Science Foundation (Basic research plan, 18JCJQJC47600 and 19JCQNJC02200).

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Authors and Affiliations



D.Y. supervised the projects; C.Y. and J.T. designed and conducted the experiments; C.Y., R.Z. and J.T. analyzed the data; C.Y., R.Z., J.T. and D.Y. wrote the manuscript.

Corresponding author

Correspondence to Dayong Yang.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key references using this protocol

Tang, J. et al. Angew. Chem. Int. Ed. Engl. 132, 2511–2516 (2020):

Yao, C. et al. J. Am. Chem. Soc. 142, 3422–3429 (2020):

Lee, J. et al. Nat. Nanotechnol. 7, 816–820 (2012):

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Yao, C., Zhang, R., Tang, J. et al. Rolling circle amplification (RCA)-based DNA hydrogel. Nat Protoc 16, 5460–5483 (2021).

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