Plant bioengineering may generate high yielding and stress-resistant crops amidst a changing climate and a growing global population. However, delivery of biomolecules to plants relies on Agrobacteriuminfection, biolistic particle delivery, the first of which is only amenable to DNA delivery. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells, however, nanostructure-mediated delivery remains unexplored for plant systems in which the dominant barrier is the plant cell wall. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver small interfering RNAs (siRNAs), and effectively silence a constitutively-expressed GFP gene in transgenic mGFP5 Nicotiana benthamiana(Nb) leaves. Leveraging the programmability of DNA nanostructures, we designed a set of DNA nanostructures of varying size, shape, compactness, and stiffness, and loaded each nanostructure with siRNA at unique RNA-attachment loci. Our results indicate that certain DNA nanostructures enable efficient plant cell internalization and delivery of siRNA to mature plant leaves. We tracked DNA nanostructure internalization into the plant cell cytoplasm of several plant species (Nb,Nicotiana tabacum, Eruca sativa, and Nasturtium officinale) and found that stiffness and size are important design elements for nanostructure internalization into plant cells. DNA nanostructures with sizes below ~10 nm and higher stiffness or compactness showed higher cellular internalization – although size or stiffness alone are not mutually-exclusive contributors. We further confirmed that siRNA loading on DNA nanostructures protects the siRNA from nuclease degradation inside plant cells, compared to free siRNA. Next, we show we can load DNA nanostructures with siRNA targeting a GFP gene to enable gene silencing in plant leaves, whereby we observe gene silencing efficiencies that match nanostructure internalization trends. Our study confirms DNA nanostructures can serve as effective scaffolds and nanoscale vehicles for siRNA delivery to plants for efficient gene silencing. This work establishes DNA nanostructures as a programmable toolset for the delivery of exogenous biomolecules to plants, and establishes guidelines for the design of DNA nanostructures for effective uptake into plant cells for various applications in plant biotechnology.

DNA nanostructures coordinate gene silencing in mature plants

2019 Fall National Meeting

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