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A dynamic DNA tetrahedron framework for active targeting

Nature Protocols volume 18, pages 1028–1055 (2023)Cite this article

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Abstract

An active targeting strategy-enabled DNA tetrahedron delivery vehicle could facilitate stable drug encapsulation and stimuli-responsive on-demand release, building a universal platform for different drug delivery requirements. Owing to the excellent biocompatible nature, programmability and remarkable cell and tissue permeability, the tetrahedral DNA nanostructure (TDN) has proven its value in the delivery of various bioactive molecules. We previously described this as a static multifunctional complex in our earlier protocol. However, static structures and passive targeting behavior might introduce off-target effects under complicated biological conditions. Therefore, in this Protocol Extension, we present a major update of the TDN delivery vehicle enabling an active targeting strategy to be used for stimuli-sensitive conformation changes and on-site cargo release, which could avoid drawbacks, including complex and time-consuming fabrication processes and undetermined cell penetration ability of other DNA-based delivery vehicles. Upon exquisite design of TDN size based on cargo type, one-pot annealing is applied to fabricate the Tiamat-designed TDN exoskeleton. Then the design of the dynamic DNA apparatus can be based on the target and environmental stimuli, including DNA strand hybridization-based and pH-sensitive DNA apparatus, and careful titration of strand lengths and mismatches is achieved using polyacrylamide and agarose gel electrophoresis, or fluorophore modifications. Finally, cargo loading strategies are designed, including site and stand titration and cargo encapsulation verification. The dynamic structures show promising targetability and effectiveness in antitumor and anti-inflammatory treatment in vitro and in vivo. Assembly and characterization in the lab takes ~5 d, and the timing for the verification of biostability and biological applications depends on the uses.

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Fig. 1: The design of the DNA tetrahedron capable of active targeting.
Fig. 2: TDN exoskeleton design and functional modifications.
Fig. 3: Illustration of two dynamic DNA apparatus candidates.
Fig. 4: Titration of pH-sensitive DNA apparatus design.
Fig. 5: Peptide loading design: loading site titration.
Fig. 6: Nucleic acid drug loading design.

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Data availability

The main data supporting the examples of this protocol are available in the supporting primary research papers17,18 Additional data can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

This study was supported by the National Key R&D Program of China (2019YFA0110600), National Natural Science Foundation of China (81970916, 81671031, 81800947 and 82101077), Sichuan Science and Technology Program (2022JDTD0021 and 2022JDRC0143) and Postdoctoral Science Foundation of China (2020T130443, 2021M702331, 2021TQ0224 and 2021M692271), and Research Funding from West China School/Hospital of Stomatology Sichuan University (RCDWJS2021-20 and RCDWJS2022-12).

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  1. These authors contributed equally: Taoran Tian, Tao Zhang, Sirong Shi.

Authors and Affiliations

  1. State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, P. R. China

    Taoran Tian, Tao Zhang, Sirong Shi, Yang Gao, Xiaoxiao Cai & Yunfeng Lin

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Contributions

Y.L. supervised and conceived of the study. T.T., Y.G., S.S. and T.Z. designed the TDN-based delivery system and completed the corresponding experiments. T.Z., T.T., S.S., X.C. and Y. G. prepared the manuscript.

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Correspondence to Yunfeng Lin.

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Nature Protocols thanks Dhiraj Bhatia, Chunhai Fan and Bin He for their contribution to the peer review of this work.

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Key references using this protocol

Tian, T. T. et al. Adv. Funct. Mater. 31, 2007342 (2021): https://doi.org/10.1002/adfm.202007342

Gao, Y. et al. Adv. Mater. 34, 2201731 (2022): https://doi.org/10.1002/adma.202201731

This protocol is an extension to: Nat. Protoc. 15, 2728–2757 (2020): https://doi.org/10.1038/s41596-020-0355-z

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Tian, T., Zhang, T., Shi, S. et al. A dynamic DNA tetrahedron framework for active targeting. Nat Protoc 18, 1028–1055 (2023). https://doi.org/10.1038/s41596-022-00791-7

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