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Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment


Although organic nanomaterials and inorganic nanoparticles possess inherent flexibility, facilitating functional modification, increased intracellular uptake and controllable drug release, their underlying cytotoxicity and lack of specificity still cause safety concerns. Owing to their merits, which include natural biocompatibility, structural stability, unsurpassed programmability, ease of internalization and editable functionality, tetrahedral DNA nanostructures show promising potential as an alternative vehicle for drug delivery and biomedical treatment. Here, we describe the design, fabrication, purification, characterization and potential biomedical applications of a self-assembling tetrahedral DNA nanostructure (TDN)–based multifunctional delivery system. First, relying on Watson-Crick base pairing, four single DNA strands form a simple and typical pyramid structure via one hybridization step. Then, the protocol details four different modification approaches, including replacing a short sequence of a single DNA strand by an antisense peptide nucleic acid, appending an aptamer to the vertex, direct incubation with small-molecular-weight drugs such as paclitaxel and wogonin and coating with protective agents such as cationic polymers. These modified TDN-based complexes promote the intracellular uptake and biostability of the delivered molecules, and show promise in the fields of targeted therapy, antibacterial and anticancer treatment and tissue regeneration. The entire duration of assembly and characterization depends on the cargo type and modification method, which takes from 2 h to 3 d.

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Fig. 1: Schematic illustration of tetrahedral DNA nanostructure-based delivery systems and their biomedical applications.
Fig. 2: Illustration of the formation of TDNs, asPNA-TDNs and aptamer-modified TDNs.
Fig. 3: Characterization of asPNA-TDNs.
Fig. 4: Characterization of aptamer-modified TDNs.
Fig. 5: Characterization of small-molecular-weight drug-loading TDNs.
Fig. 6: Characterization of PEI/TDNs and PEGylated-protamine/TDNs.
Fig. 7: Biostability analysis of TDNs and aptamer-modified TDNs.
Fig. 8: Bacterial uptake of asPNA-TDNs.
Fig. 9: Aptamer-modified TDNs for cell targeting.
Fig. 10: In vitro biomedical effects of TDN-based delivery complexes.
Fig. 11: In vivo biomedical applications of TDNs and TDN-based delivery complexes.

Data availability

All data generated in this study can be obtained from the corresponding author upon reasonable request.


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This study was supported by the National Key R&D Program of China (2019YFA0110600) and the National Natural Science Foundation of China (81970916, 81671031 and 81800947).

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Authors and Affiliations



Y.L. supervised and conceived the research. T.T., W.M., Y.Z., N.L., S.S., Q.L., X.X., Q.Z., S.L., M.L. and Y.G. designed the TDN-based delivery systems and completed the corresponding experiments. T.Z., T.T., S.L., R.Z., X.C. and Y.L. interpreted data and wrote the manuscript.

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Correspondence to Yunfeng Lin.

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Peer review information Nature Protocols thanks Yonggang Ke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Ma, W. et al. Nano Lett. 19, 4505–4517 (2019):

Zhang, Y. et al. Nano Lett. 18, 5652–5659 (2018):

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Zhang, T., Tian, T., Zhou, R. et al. Design, fabrication and applications of tetrahedral DNA nanostructure-based multifunctional complexes in drug delivery and biomedical treatment. Nat Protoc 15, 2728–2757 (2020).

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