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Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers

Nature Protocols volume 10, pages 1508–1524 (2015)Cite this article

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Abstract

We describe a comprehensive protocol for the preparation of multifunctional DNA nanostructures termed nanoflowers (NFs), which are self-assembled from long DNA building blocks generated via rolling-circle replication (RCR) of a designed template. NF assembly is driven by liquid crystallization and dense packaging of building blocks, which eliminates the need for conventional Watson-Crick base pairing. As a result of dense DNA packaging, NFs are resistant to nuclease degradation, denaturation or dissociation at extremely low concentrations. By manually changing the template sequence, many different functional moieties including aptamers, bioimaging agents and drug-loading sites could be easily integrated into NF particles, making NFs ideal candidates for a variety of applications in biomedicine. In this protocol, the preparation of multifunctional DNA NFs with highly tunable sizes is described for applications in cell targeting, intracellular imaging and drug delivery. Preparation and characterization of functional DNA NFs takes ∼5 d; the following biomedical applications take ∼10 d.

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Figure 1: Schematic illustration of noncanonical self-assembly and biomedical applications of multifunctional DNA NFs.
Figure 2: Designing the RCR template and primer for DNA NF preparation.
Figure 3: SEM imaging of the morphology and size of NFs during the growth process with reaction time from 0.5 to 30 h.
Figure 4: Bioimaging of intracellular behavior of NFs and Dox delivered by NFs.

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Acknowledgements

This work was supported by the National Key Scientific Program of China (2011CB911000), National Natural Science Foundation of China (NSFC) grants (21325520, 21327009, 21221003, J1210040, 21177036 and 21135001) and the China National Instrumentation Program 2011YQ03012412. It is also supported by the US National Institutes of Health (GM079359 and CA133086).

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Authors and Affiliations

  1. Molecular Science and Biomedicine Laboratory, State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center of Molecular Engineering for Theranostics, Hunan University, Changsha, China

    Yifan Lv, Rong Hu, Guizhi Zhu, Xiaobing Zhang, Lei Mei, Qiaoling Liu, Liping Qiu, Cuichen Wu & Weihong Tan

  2. Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at Bio/Nano Interface, Shands Cancer Center, University of Florida Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida, USA

    Rong Hu, Guizhi Zhu, Liping Qiu, Cuichen Wu & Weihong Tan

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Contributions

G.Z., R.H., X.Z. and W.T. originated the method of preparing DNA NFs; Y.L., G.Z., R.H., X.Z., L.M., Q.L., L.Q., C.W. and W.T. were responsible for the content of the protocol. Y.L. and W.T. wrote the manuscript, and all authors contributed to the revision of the manuscript.

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Correspondence to Xiaobing Zhang or Weihong Tan.

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Integrated supplementary information

Supplementary Figure 1 Predicted secondary structures of the linear RCR template and elongated concatamer RCR product.

(a) The linear RCR template is predicted to have a 3-way junction secondary structure. Sequence of template is shown in Table 1. (b) The elongated concatamer RCR product is predicted to have branched aptamer structures protruding and aligning on alternative sides and many dsDNA (for drug loading) on the backbone and stem. Structures were predicted using the Nupack software.

Supplementary Figure 2 Electrophoresis and confocal fluorescence microscopy characterization of NFs.

a) Agarose gel (2%) electrophoresis image indicating the elongation of DNA through RCR. Lane M: 20 bp DNA marker, lane 1: template, lane 2: primer, lane 3: template and primer after T4 DNA ligase treatment. The tailing results from the complicated structures formed through intra- and intermolecular base pairs. Lane 4: RCR product after phi29 DNA polymerase treatment. Polymerization of dNTP monomers and dense packaging-driven assembly results in huge molecular weight of RCR products; therefore, the corresponding band shows no migration. Hu, R. et al. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery. Angew. Chem. Int. Ed. 2014. Volume 53. Pages 5821–5826. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission. b) Confocal fluorescence microscopy imaging of Cy5-dUTP-integrated NFs. The fluorescence signal (Red) of Cy5 (maximum excitation wavelength is 650 nm and maximum emission wavelength is 662 nm) is obvious and indicates the successful polymerization of Cy5-dUTP and the normal function of Cy5. NFs with a size of 2 μm (RCR for 20-30 h) are used for this imaging. Scale bar, 5 μm. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), 16438–16445. Copyright 2013 American Chemical Society.

Supplementary Figure 3 Characterizations of NFs.

a) Dynamic light scattering measurement of NFs. DLS data reveals the size distribution of NFs and the average radius is calculated to be about 150 nm. NFs with 15 h of RCR reaction time are used in this experiment. Hu, R. et al. DNA nanoflowers for multiplexed cellular imaging and traceable targeted drug delivery. Angew. Chem. Int. Ed. 2014. Volume 53. Pages 5821–5826. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission. b) TEM images of NFs displays ultrathin sheet sections (indicated by arrows). c) Enlarged partial view of the dashed box. These results provide clear evidence of the internal hierarchical structures and the dense DNA packaging in NFs. NFs with 10 h of RCR reaction time are used for this experiment. Adapted from ref. 31. d) AFM imaging of NFs. e) Cross-sectional plot indicated with the white line. AFM imaging displays that the NFs are monodisperse with a size of 100-200 nm. NFs with 10 h of RCR reaction time are used for this experiment. Adapted from ref. 31. f) Bright field. g) Polarized light imaging of (f). When exposed between crossed polarizers, NFs displayed disc-shaped optical textures as a result of the birefringence of liquid crystalline structure, which is an optical property of anisotropic materials. This is a direct demonstration of liquid crystalline structures of NFs. NFs with 10-15 h of RCR reaction time are used for this experiment. Panels b–g adapted with permission from J. Am. Chem. Soc. 2013, 135(44), 16438–16445. Copyright 2013 American Chemical Society.

Supplementary Figure 4 Exceptional stability of NFs.

(a−c) SEM images of NFs treated with DNase I (a, b, 5 U/mL) and human serum (c, 10% diluted) for 24 h at 37 °C. (d−f) SEM images displaying NFs heated at 170 °C for 0.5 h (d), treated with urea (5 M) for 0.5 h (e), and diluted 100 times (f). The maintenance of NF structural integrity indicates the high stability of NFs. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), 16438–16445. Copyright 2013 American Chemical Society.

Supplementary Figure 5 Flow cytometry results demonstrating the selective recognition of sgc8c-incorporated NFs.

(a) CEM cells (target cell line, suspension); dark green=cell only, light green=cells incubated with NFs. (b) Ramos cells (nontarget cell line, suspension); dark green=cell only, light green=cells incubated with NFs. (c) HeLa cells (target cell line, adherent); gray=cell only, red=cells incubated with NFs. Target cell lines show an obvious fluorescence signal shift compared with nontarget cell line, which demonstrates a high selectivity of NFs toward target cells. These results show that the conjugated aptamer preserves its binding affinity and specificity. NFs with a size of 200~300 nm (RCR for 10-15 h) are used for this experiment. Angew. Chem. Int. Ed. 2014. Volume 53. Pages 5821–5826. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Adapted with permission.

Supplementary Figure 6 Drug-loading into the preferable drug-associated sequences of NFs and the cytotoxicity of NFs toward target/nontarget cells.

(a) Drug-loading into the preferable drug-associated sequences of NFs. Left: NFs are incubated with Dox in Dulbecco’s PBS at room temperature. The whole mixture shows uniform red, which is the color of Dox solution. Right: After incubation for 24 hours, the mixture is centrifuged at 14,000 g for 5 min. The supernatant turns light upon losing part of the Dox, while the NF concentrate turns dark red upon Dox loading. Both observation with the naked eye and the following ultraviolet absorption measurement demonstrate the drug-loading ability of DNA NFs. NFs with a size of 200~300 nm (RCR for 10-15 h) are used for this experiment. (b) The correspondence between NF concentration and cell viability. Red line, cell viability of target CEM cells with increasing NF concentration; black line, cell viability of nonarget Ramos cells with increasing NF concentration. No significant cytotoxicity of NFs toward either target or nontarget cells is observed in panel a, which indicates the satisfactory biocompatibility of NFs. (c) SEM images of NFs used in this cytotoxicity experiment. Scale bar: 50 μm. Inset panel, high-resolution imaging of a single NF. Scale bar: 300 nm. NFs with a size of 200~300 nm (RCR for 10-15 h) are used for this experiment. Panels b and c adapted with permission from J. Am. Chem. Soc. 2013, 135(44), 16438–16445. Copyright 2013 American Chemical Society.

Supplementary Figure 7 MTS assay results show selective cytotoxicity of Dox delivered by NFs.

(a) Cell viability of target HeLa cells with NF-Dox complex or free Dox treatment. (b) Cell viability of target CEM cells with NF-Dox complex or free Dox treatment. (c) Cell viability of nontarget Ramos cells with NF-Dox complex or free Dox treatment. Red triangle, cytotoxicity of free Dox; black dots, cytotoxicity of NF-Dox complex. Free Dox shows nonselectivity toward both target (HeLa and CEM cells) and nontarget (Ramos cells) cell lines, while NF-Dox complex only shows cytotoxicity toward target cell lines (HeLa and CEM cells). In contrast to nonselective cytotoxicity of free Dox in both target cells and nontarget cells, the selective cytotoxicity of Dox delivered by NFs indicates their ability to perform targeted drug delivery. NFs with a size of 200~300 nm (RCR for 10-15 h) were used for this experiment. Adapted with permission from J. Am. Chem. Soc. 2013, 135(44), 16438–16445. Copyright 2013 American Chemical Society.

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Lv, Y., Hu, R., Zhu, G. et al. Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers. Nat Protoc 10, 1508–1524 (2015). https://doi.org/10.1038/nprot.2015.078

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