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Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications

Nature Protocols (2025)Cite this article

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Abstract

Artificial DNA nanostructures, with their sequence programmability, precise molecular recognition and tunable stimuli responsiveness, bridge material chemistry and biomedicine. Here we detail the design and construction of hybridization chain reaction (HCR)-based DNA nanoframeworks, a class of DNA nanostructures with programmable sequences and customizable functions. HCR is an efficient, enzyme-free amplification strategy that isothermally produces nicked double-stranded DNA with periodically repeated modules via the assembly of two DNA hairpins, triggered by a DNA initiator. In contrast to other available assembly methods for the synthesis of DNA nanostructures, such as tile-mediated assembly, DNA origami and rolling circle amplification, the HCR method offers improved stability and efficiency under mild conditions, without reliance on enzymatic activity. The procedure uses radical polymerization to integrate DNA initiator into nanoframeworks, with overhangs complementary to functional sequences — termed linkers —which are amplified and incorporated through HCR. The linkers enable the incorporation of functional nucleic acid sequences. The HCR-based DNA nanoframeworks facilitate the loading capability of the delivered molecules, showing notable therapeutic efficacy and biosensing sensitivity. Preparation time for HCR-based DNA nanoframeworks ranges from 30 h to 45 h, depending on the payload.

Key points

  • The procedure covers the design and preparation of the nanoframeworks, the loading of functional nucleic acid sequences via hybridization chain reaction and their characterization, followed by their evaluation as therapeutics and as biosensors.

  • Th procedure uses an enzyme-free isothermal amplification-based self-assembly approach with mild reaction conditions and high amplification efficiency, which is easy to implement.

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Fig. 1: Schematic illustration of HCR-based DNA nanoframeworks for therapeutic and biosensing applications.
Fig. 2: Oligonucleotides loading (miRNA/siRNA) for gene silencing.
Fig. 3: Cas9 RNP loading for gene editing.
Fig. 4: mRNA loading for mRNA-based therapeutics.
Fig. 5: The controlled drug release of DNA nanoframeworks.
Fig. 6: DNA nanoframeworks enabling lysosome biosensing.
Fig. 7: DNA nanoframeworks enabling mitochondria biosensing.
Fig. 8: Characterization of HCR-based DNA nanoframework.

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Data availability

The main data supporting the examples of this protocol are available in the supporting primary research papers (https://doi.org/10.1038/s41467-021-21442-7, https://doi.org/10.1126/sciadv.adi3602, https://doi.org/10.1002/adma.202300823, https://doi.org/10.1002/anie.202207770, https://doi.org/10.1002/adfm.202312880). Additional data are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (grant nos. 22225505, 22322407 and 22174097). D.Y. thanks Fudan University Ruiqing Education Funding.

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Authors and Affiliations

  1. Department of Chemistry, State Key Laboratory of Molecular Engineering of Polymers, Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, College of Chemistry and Materials, Fudan University, Shanghai, P.R. China

    Zhaoyue Lv, Peiran Li, Mingxing Liu & Dayong Yang

  2. State Key Laboratory of Synthetic Biology, Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin, P.R. China

    Zhaoyue Lv, Peiran Li, Mingxing Liu, Chi Yao & Dayong Yang

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  1. Zhaoyue Lv
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  2. Peiran Li
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Contributions

D.Y. and C.Y. supervised the projects. Z.L. and P.L. designed and conducted the experiments. Z.L., P.L. and M.L analyzed the data. Z.L., P.L., M.L, C.Y. and D.Y. wrote the manuscript.

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Correspondence to Chi Yao or Dayong Yang.

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Nature Protocols thanks Jong Bum Lee, Veikko Linko, Chengde Mao, Simona Ranallo and Olavi Reinsalu for their contribution to the peer review of this work.

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Key references

Li, F. et al. Nat. Commun. 12, 1138 (2021): https://doi.org/10.1038/s41467-021-21442-7

Song, N. et al. Sci. Adv. 9, eadi3602 (2023): https://doi.org/10.1126/sciadv.adi3602

Lv, Z. et al. Adv. Mater. 35, e2300823 (2023): https://doi.org/10.1002/adma.202300823

Dong, Y. et al. Angew. Chem. Int. Ed. 61, e202207770 (2022): https://doi.org/10.1002/anie.202207770

Yuan, M. et al. Adv. Funct. Mater. 34, 2312880 (2023): https://doi.org/10.1002/adfm.202312880

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Lv, Z., Li, P., Liu, M. et al. Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01183-3

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