Nanotubes effectively deliver siRNA to intact plant cells and protect siRNA against nuclease degradation

Abstract

Post-transcriptional gene silencing (PTGS) is a powerful tool to understand and control plant metabolic pathways, which is central to plant biotechnology. PTGS is commonly accomplished through delivery of small interfering RNA (siRNA) into cells. While siRNA delivery has been optimized for mammalian systems, it remains a significant challenge for plants due to the plant cell wall. Standard plant siRNA delivery methods (Agrobacterium and viruses) involve coding siRNA into DNA vectors, and are only tractable for certain plant species. Herein, we develop a nanotube-based platform for direct delivery of siRNA, and show high silencing efficiency in intact plant cells. We demonstrate that nanotubes successfully deliver siRNA and silence endogenous genes owing to effective intracellular delivery and nanotube-induced protection of siRNA from nuclease degradation. This study establishes that nanotubes, which are below the size exclusion limit of the plant cell wall, could enable a myriad of plant biotechnology applications that rely on RNA delivery. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/564427 doi: bioRxiv preprint Plants are central in providing over 25% of our most clinically-relevant drugs, are at the core of our sustainability efforts, and will benefit from genetic engineering to feed our growing population in the midst of climate change. Plant biotechnology is currently limited by the cost, ease, and throughput of methods for probing plant genetics and gene expression profiles. Consequently, less than a dozen complete biosynthetic pathways are known for plant natural products that have been reconstituted heterologously, compared to the ~1000 known biosynthetic pathways in bacteria and fungi 1 . RNA interference (RNAi) is sequence-specific inhibition of gene expression at the messenger RNA (mRNA) level, and can either consist of transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS). In PTGS, small RNA moleculesmicro (miRNA) or small interfering (siRNA)direct enzyme complexes to degrade mRNA molecules, hence suppress their activity by preventing translation.PTGS has shown to be a prominent tool in plants for genotype-phenotype mapping 2 , discovery of new biosynthetic pathways 3 , increased production of valuable small molecules 4,5 , understanding the functions of genes and proteins 6 , and to confer resistance to plant diseases 7,8 . One common way of utilizing PTGS in plants is to directly deliver siRNA molecules into cells. However, plants have a cell wall which presents a barrier to exogenous biomolecule delivery, whereby the plant cell wall size exclusion limit is ~ 5-20 nm 9 . Consequently, viral vectors combined with Agrobacterium tumefaciens delivery is the preferred method to deliver siRNA into intact plant cells. Viral vectors present the advantage of directly and strongly expressing the siRNA without relying on plant transformation, however, mos...

Nanoparticle‐mediated gene transformation strategies for plant genetic engineering

“…The SWCNTs serve as gene-delivery vehicles that can penetrate the membranes of organelles and have many advantages in plant genetic engineering (Kwak et al, 2019), such as biocompatibility, high aspect ratio, high surface area to volume ratio and exceptional tensile strength (Hendler-Neumark and Bisker, 2019;Mohanta et al, 2019). As a result of their high surface area to volume ratio, SWCNTs can be loaded with large quantities of DNA for transfer into plant cells (Burlaka et al, 2015;Kwak et al, 2019;Demirer et al, 2019aDemirer et al, , 2019bDemirer et al, , 2019c 2019c). Finally, the cargo-nanoparticle complexes formed by SWCNTs and their loaded cargoes have strong intrinsic near-infrared (nIR) fluorescence, allowing them to be tracked in real time (Li et al, 2014;Hendler-Neumark and Bisker, 2019).…”

“…Although nanoparticles have many merits in plant science, such as the modulation of plant growth, nanobiosensors and gene carriers (Zhao et al, 2017;Kwak et al, 2019;Lee et al, 2019;Thapa et al, 2019;Demirer et al, 2019aDemirer et al, , 2019bDemirer et al, , 2019c, there are still many challenges in plant gene transformation.…”

“…buffer conditions, DNA/nanoparticle ratio, exposure time and sterility) for delivery to different tissues (e.g. pollen, callus, embryonic tissue and germ-line cells) (Demirer et al, 2019a(Demirer et al, , 2019b(Demirer et al, , 2019c.…”

“…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).…”