Abstract
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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Bao, M., Xie, J. & Huck, W. T. S. Recent advances in engineering the stem cell microniche in 3D. Adv. Sci. 5, 1800448 (2018).
Chaudhuri, O. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat. Mater. 15, 326–334 (2016).
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).
Baker, B. M. et al. Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments. Nat. Mater. 14, 1262–1268 (2015).
Zhang, Y. S. & Khademhosseini, A. Advances in engineering hydrogels. Science 356, eaaf3627 (2017).
Yuk, H., Lu, B. & Zhao, X. Hydrogel bioelectronics. Chem. Soc. Rev. 48, 1642–1667 (2019).
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).
Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).
Yang, D. et al. DNA materials: bridging nanotechnology and biotechnology. Acc. Chem. Res. 47, 1902–1911 (2014).
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).
Wang, D., Hu, Y., Liu, P. & Luo, D. Bioresponsive DNA hydrogels: beyond the conventional stimuli responsiveness. Acc. Chem. Res. 50, 733–739 (2017).
Shao, Y., Jia, H., Cao, T. & Liu, D. Supramolecular hydrogels based on DNA self-assembly. Acc. Chem. Res. 50, 659–668 (2017).
Kahn, J. S., Hu, Y. & Willner, I. Stimuli-responsive DNA-based hydrogels: from basic principles to applications. Acc. Chem. Res. 50, 680–690 (2017).
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).
Wang, J. et al. Clamped hybridization chain reactions for the self-assembly of patterned DNA hydrogels. Angew. Chem. Int. Ed. 56, 2171–2175 (2017).
Nöll, T. et al. Construction of three-dimensional DNA hydrogels from linear building blocks. Angew. Chem. Int. Ed. 53, 8328–8332 (2014).
Li, J. et al. Functional nucleic acid-based hydrogels for bioanalytical and biomedical applications. Chem. Soc. Rev. 45, 1410–1431 (2016).
English, M. A. et al. Programmable crispr-responsive smart materials. Science 365, 780–785 (2019).
Li, F., Tang, J., Geng, J., Luo, D. & Yang, D. Polymeric DNA hydrogel: Design, synthesis and applications. Prog. Polym. Sci. 98, 101163 (2019).
Um, S. H. et al. Enzyme-catalysed assembly of DNA hydrogel. Nat. Mater. 5, 797–801 (2006).
Cheng, E. et al. A pH-triggered, fast-responding DNA hydrogel. Angew. Chem. Int. Ed. 48, 7660–7663 (2009).
Lee, J. B. et al. A mechanical metamaterial made from a DNA hydrogel. Nat. Nanotechnol. 7, 816–820 (2012).
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).
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).
Geng, J. et al. A fluorescent biofunctional DNA hydrogel prepared by enzymatic polymerization. Adv. Healthc. Mater. 7, 1700998 (2018).
Merindol, R., Delechiave, G., Heinen, L., Catalani, L. H. & Walther, A. Modular design of programmable mechanofluorescent DNA hydrogels. Nat. Commun. 10, 528 (2019).
Hamada, S. et al. Dynamic DNA material with emergent locomotion behavior powered by artificial metabolism. Sci. Robot. 4, eaaw3512 (2019).
Madl, C. M., Heilshorn, S. C. & Blau, H. M. Bioengineering strategies to accelerate stem cell therapeutics. Nature 557, 335–342 (2018).
De Luca, M. et al. Advances in stem cell research and therapeutic development. Nat. Cell Biol. 21, 801–811 (2019).
Qian, X. et al. Untethered recyclable tubular actuators with versatile locomotion for soft continuum robots. Adv. Mater., e1801103 (2018).
Wang, C. et al. Soft ultrathin electronics innervated adaptive fully soft robots. Adv. Mater. 30, e1706695 (2018).
Cangialosi, A. et al. DNA sequence-directed shape change of photopatterned hydrogels via high-degree swelling. Science 357, 1126–1130 (2017).
Yang, C. & Suo, Z. Hydrogel ionotronics. Nat. Rev. Mater. 3, 125–142 (2018).
Gao, T. et al. Design and fabrication of flexible DNA polymer cocoons to encapsulate live cells. Nat. Commun. 10, 2946 (2019).
Ye, D. et al. Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction. Nat. Protoc. 15, 2163–2185 (2020).
Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).
Li, L. et al. Nucleic acid aptamers for molecular diagnostics and therapeutics: Advances and perspectives. Angew. Chem. Int. Ed. Engl. 60, 2221–2231 (2021).
McCarthy, S. A., Davies, G. L. & Gun’ko, Y. K. Preparation of multifunctional nanoparticles and their assemblies. Nat. Protoc. 7, 1677–1693 (2012).
Acknowledgements
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).
Author information
Authors and Affiliations
Contributions
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
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Nature Protocols thanks the anonymous reviewers 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.
Related links
Key references using this protocol
Tang, J. et al. Angew. Chem. Int. Ed. Engl. 132, 2511–2516 (2020): https://doi.org/10.1002/anie.201913549
Yao, C. et al. J. Am. Chem. Soc. 142, 3422–3429 (2020): https://doi.org/10.1021/jacs.9b11001
Lee, J. et al. Nat. Nanotechnol. 7, 816–820 (2012): https://doi.org/10.1038/NNANO.2012.211
Supplementary information
Supplementary Data 1
CAD file for 3D printing design
Rights and permissions
About this article
Cite this article
Yao, C., Zhang, R., Tang, J. et al. Rolling circle amplification (RCA)-based DNA hydrogel. Nat Protoc 16, 5460–5483 (2021). https://doi.org/10.1038/s41596-021-00621-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41596-021-00621-2
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.
