Targeted downregulation of select endogenous plant genes is known to confer disease or pest resistance in crops and is routinely accomplished via transgenic modification of plants for constitutive gene silencing. An attractive alternative to the use of transgenics or pesticides in agriculture is the use of a ‘green’ alternative known as RNAi, which involves the delivery of siRNAs that downregulate endogenous genes to confer resistance. However, siRNA is a molecule that is highly susceptible to enzymatic degradation and is difficult to deliver across the lignin-rich and multi-layered plant cell wall that poses the dominant physical barrier to biomolecule delivery in plants. We have demonstrated that DNA nanostructures can be utilized as a cargo carrier for direct siRNA delivery and gene silencing in mature plants. The size, shape, compactness and stiffness of the DNA nanostructure affect both internalization into plant cells and subsequent gene silencing efficiency. Herein, we provide a detailed protocol that can be readily adopted with standard biology benchtop equipment to generate geometrically optimized DNA nanostructures for transgene-free and force-independent siRNA delivery and gene silencing in mature plants. We further discuss how such DNA nanostructures can be rationally designed to efficiently enter plant cells and deliver cargoes to mature plants, and provide guidance for DNA nanostructure characterization, storage and use. The protocol described herein can be completed in 4 d.
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All materials are available from commercial sources or can be derived using methods described in this study. All data and controls relevant to the protocol have been included in the Supplementary Information. Raw data such as unprocessed image files can be obtained from the corresponding author upon reasonable request.
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Hu. Z. acknowledges the support of the Chinese National Natural Science Foundation (21605153). The authors acknowledge support from a Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Stanley Fahn PDF Junior Faculty Grant under award no. PF-JFA-1760, a Beckman Foundation Young Investigator Award, a USDA AFRI award, a grant from the Gordon and Betty Moore Foundation, a USDA NIFA award, a USDA-BBT EAGER award, support from the Chan-Zuckerberg Foundation and an FFAR New Innovator Award (to M.P.L.). G.S.D. is supported by a Schlumberger Foundation Faculty for the Future Fellowship. The authors also acknowledge support from UC Berkeley Molecular Imaging Center (supported by the Gordon and Betty Moore Foundation), the QB3 Shared Stem Cell Facility and the Innovative Genomics Institute (IGI).
The authors declare no competing interests.
Peer review information Nature Protocols thanks Veikko Linko 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
Zhang, H. et al. Proc. Natl Acad. Sci. USA 116, 7543–7548 (2019): https://doi.org/10.1073/pnas.1818290116
Zhang, H. et al. Nat. Commun. 10, 1006 (2019): https://doi.org/10.1038/s41467-019-09004-4
Integrated supplementary information
Supplementary Figure 1 Representative gel image for characterization of the formation of DNA nanostructures.
a,b,10% native PAGE gel showing the formation of tetrahedron (a) and DHT monomer A (b). Lane1: marker; lane 2: DNA nanostructure.
Introduce a tiny puncture into the Nb plant leaf with a pipette tip (left) on the leaf abaxial surface. Center the syringe tip at the puncture area (middle), and gently push the syringe plunger until all the liquid is infiltrated (right).
a, Representative confocal images of mGFP5 Nb leaves. Scale bar: 100 µm. b, To validate GFP expression in mGFP5 Nb, a western gel shows the correct GFP band size (~27 kDa) for GFP extracted from mGFP5 Nb leaves.
Supplementary Figure 4 Internalization of Cy3-labeled DHT monomer into three different plant species.
Cy3-labeled HT monomer can internalize into tobacco, arugula and watercress leaf cells. Scale bars, 100 µm.
Supplementary Figure 5 Representative confocal images to test the internalization mechanism of the Cy3-labeled DHT monomer into Nb leaves.
a, Temperature dependence of internalization: we observe internalization predominately when plants infiltrated with Cy3-DHT monomers are incubated at 21 °C, but not when incubated at 4 °C, suggesting that the internalization process is energy dependent. Scale bars, 50 µm. b, Leaves pretreated with 33 µM wortmannin (incubated for half an hour before DNA nanostructure infiltration), a chemical inhibitor of endocytosis, show greatly reduced Cy3-DHT nanostructure internalization compared with non-treated counterparts, suggesting that the nanostructures enter the plant cells through receptor-mediated endocytosis.
a, Representative confocal images of mGFP5 Nb leaves 7 d post-infiltration with PBS (control), siRNA–tetrahedron nanostructures, or siRNA–DHT monomer nanostructures, showing GFP fluorescence recovery. Scale bars, 100 µm. b, Quantitative fluorescence intensity analysis of confocal images. n.s.= non significant (s.d., n = 15). c, Statistical analysis and representative western gel of GFP extracted from nanostructure-treated leaves 72 h post-infiltration. n.s.= non significant (s.d., n = 3).
a, qPCR of leaves infiltrated with free siRNA, siRNA-loaded nanostring, DHT-c, tetrahedron or DHT-s 1d post-infiltration. ****P < 0.0001 in one-way ANOVA. Error is SEM (n = 4). b, Scheme showing the proposed silencing pathways induced by different siRNA-loaded DNA nanostructures.
qPCR results from GFP5 Nb leaves infiltrated with DHT monomer alone 2 d post-infiltration showing no mRNA change. Control samples are non-infiltrated leaves. Error bars indicate s.e.m. (n = 4).
Supplementary Figure 9 DNA nanostructure-infiltrated Nb leaves show no stress response as measured by qPCR.
qPCR analysis of NbrbohB, a known plant stress gene, to test the toxicity of DNA nanostructures used to deliver siRNA. Nanostructures do not upregulate the plant stress gene. Control samples are PBS buffer-infiltrated leaves. Error bars indicate s.e.m. (n = 3).
Supplementary Figure 10 Silencing of endogenous functional Nb ROQ1 gene with siRNA loaded on tetrahedron DNA nanostructures.
Free ROQ1 siRNA does not induce significant silencing of the ROQ1 gene, whereas 100 nM ROQ1 siRNA loaded on the tetrahedron nanostructure yields a near 50% decrease of ROQ1 mRNA as assessed by qPCR of infiltrated Nb leaves compared with the non-treated control leaves. ** P = 0.0010 in one-way ANOVA. Error bars indicate s.e.m. (n = 3).
a, 10% native PAGE gel showing incubation of DHT monomer with plant cell lysate at different time points. Lane 1: marker; lane 2: DHT monomer only as control; lanes 3–8: DHT monomer incubated with plant cell lysate at 0 h, 12 h, 24 h, 48 h, 72 h and 96 h at room temperature. b, Normalized band intensity analysis of the gels in a where 100% band intensity is defined as the DHT monomer control alone without the plant cell lysate. Upward deviations from 100% are due to nonspecific protein adsorption to nanostructures that slightly increase the band width and optical density.
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Zhang, H., Zhang, H., Demirer, G.S. et al. Engineering DNA nanostructures for siRNA delivery in plants. Nat Protoc 15, 3064–3087 (2020). https://doi.org/10.1038/s41596-020-0370-0
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