DNA Nanostructures Coordinate Gene Silencing in Mature Plants

Abstract

SignificancePlant bioengineering will be necessary to sustain plant biology and agriculture, where the delivery of biomolecules such as DNA, RNA, or proteins to plant cells is at the crux of plant biotechnology. Here, we show that DNA nanostructures can passively internalize into plant cells and deliver small interfering RNA (siRNA) to mature plant tissues. Furthermore, we demonstrate that nanostructure size, shape, compactness, and stiffness, affect both nanostructure internalization into plant cells and subsequent gene silencing efficiency. Interestingly, we also find that the siRNA attachment locus affects the endogenous plant gene silencing pathway. Our work demonstrates programmable passive delivery of biomolecules to plants, and details the figures of merit for future implementation of DNA nanostructures in agriculture. AbstractPlant bioengineering may generate high yielding and stress-resistant crops amidst a changing climate and a growing global population (1-3). However, delivery of biomolecules to plants relies on Agrobacterium infection (4) or biolistic particle delivery (5), the former of which is only amenable to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall which is absent in mammalian cells and poses the dominant physical barrier to exogenous biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells, however, nanoparticle-mediated delivery remains unexplored for passive biomolecule delivery across the cell wall in plants. 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 constitutivelyexpressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and the corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies, but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures, and details both the design parameters of importance for plant cell internalization, and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

“…To facilitate RNA delivery inside the plant cell, RNA molecules are usually conjugated to carrier compounds, as mentioned above (Jiang et al, 2014;Numata et al, 2014). Recently, DNA nanostructures, such as 3D tetrahedron, 1D hairpin tile, and 1D nanostring, were used to facilitate the delivery and biological action of 21-nt GFP sRNAs in infiltrated N. benthamiana leaves (Zhang et al, 2019). Instead of remaining in the mesophyllic apoplast, the nanostructure-conjugated sRNAs entered the symplast and silenced GFP expression.…”

RNA‐based biocontrol compounds: current status and perspectives to reach the market

“…This will be dependent, however, on the cost of production and efficiency of the developed nanocarriers. Recently, the use of DNA nanostructures serving as RNA carriers through Watson–Crick base pairing has been reported to be an efficient tool for delivery of siRNA into plant cells . Parameters such as the compactness, stiffness, size, shape and location of the siRNA attachment locus on the nanostructure will have to be taken into consideration during formulation in order to ensure proper internalization of the nanostructure into plant cells for a corresponding good gene silencing efficiency.…”

Nanoparticle‐mediated gene transformation strategies for plant genetic engineering

“…The cell walls in plant cells may form a natural obstacle to the delivery of biological molecules such as DNA and RNA, however. Thus, Zhang et al (2019) developed DNA nanoparticles as a platform for siRNA delivery in plants. The efficiency of siRNA gene silencing was determined by the size, shape, stiffness, compactness and attachment site (siRNA attachment locus) of the designed DNA nanostructures.…”

“…When the pDNA:SWNT ratio is 1:3-1:6 (w/w), nanoparticles have a high potential to carry cargoes across the plasma membrane (Kwak et al, 2019). DNA lengths of 20-15 000 bp can be successfully delivered to plant cells (Zhao et al, 2017;Zhang et al, 2019;Demirer et al, 2019aDemirer et al, , 2019bDemirer et al, , 2019c, and the upper limit of the maximum plasmid size delivered by nanoparticles has not yet been determined.…”

“…Nanoparticles are nanoscale particles (1–100 nm, general nanoparticles; 200 nm, magnetic nanoparticles; 148–200 nm, natural peptides; 15–120 nm, clay nanoparticles; and 130–140 nm, cationic fluorescence nanoparticles) with a wide range of applications, such as in agriculture, cosmetics, biomedicine and electronics. In recent decades, many types of nanoparticles have been developed, including DNA nanoparticles, carbon‐based nanoparticles (single‐ and multiwalled carbon nanotubes), metal‐based nanoparticles (nanoaluminum, nanozinc, nanogold, TiO 2 , ZnO and Al 2 O 3 ) (Zhang et al ., 2019; Demirer et al ., 2019a, 2019b, 2019c) and composites or combined nanoparticles such as chitosan‐complexed single‐walled carbon nanotubes (Figure 1) (Kwak et al ., 2019; Demirer et al ., 2019a, 2019b, 2019c).…”