DNA nanostructures coordinate gene silencing in mature plants

Abstract

Delivery of biomolecules to plants relies on Agrobacterium infection or biolistic particle delivery, the former of which is amenable only 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 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 without external mechanical aid remains unexplored for 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 siRNAs, and effectively silence a constitutively expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and 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 both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

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.…”

“…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. 23 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. Although RNA-based biocontrol compounds are still in the research and development (R&D) pipeline, they are broadly anticipated to fall under the following categories: direct control agents, resistance factor repressors, developmental disruptors and growth enhancers.…”

Derivatized Carbon Nanotubes for Gene Therapy in Mammalian and Plant Cells

“…Nanomaterial-mediated gene transfer has already been explored for the delivery of genetic material into mammalian cells and has brought noteworthy traction among plant cell biologists as well. [76,77] Recent reports [78][79][80] have exemplified the use of CNTs as a molecular vehicle into the protoplast, [81] which can passively traverse through the cell membrane.…”

“…Conventional methods of gene‐transfer suffer from limitations such as tissue damage, inefficiency at targeting specific cell organelle and poor cellular penetration. Nanomaterial‐mediated gene transfer has already been explored for the delivery of genetic material into mammalian cells and has brought noteworthy traction among plant cell biologists as well . Recent reports have exemplified the use of CNTs as a molecular vehicle into the protoplast, which can passively traverse through the cell membrane.…”

“…Thus it is crucial to control the bending stiffness and the compactness with which the biomolecule is linked to the nanotube as well. Zhang et al . explored the stiffness of SWCNTs nanostructure that facilitates needlelike plant cell internalization in Nicotiana benthamiana .…”