Biology subjects

Goh, N. S.

Publications and source records attributed to Goh, N. S..

6 recordsLinked to original sources

bioRxiv2021

Polymer-Conjugated Carbon Nanotubes for Biomolecule Loading

Nanomaterials have emerged as an invaluable tool for the delivery of biomolecules such as DNA and RNA, with various applications in genetic engineering and post-transcriptional genetic manipulation. Alongside this development, there has been an increasing use of polymer-based techniques, such as polyethyleneimine (PEI), to electrostatically load polynucleotide cargoes onto nanomaterial carriers. However, there remains a need to assess nanomaterial properties, conjugation conditions, and biocompatibility of these nanomaterial-polymer constructs, particularly for use in plant systems. In this work, we develop mechanisms to optimize DNA loading on single-walled carbon nanotubes (SWNTs) with a library of polymer-SWNT constructs and assess DNA loading ability, polydispersity, and both chemical and colloidal stability. Counterintuitively, we demonstrate that polymer hydrolysis from nanomaterial surfaces can occur depending on polymer properties and attachment chemistries, and describe mitigation strategies against construct degradation. Given the growing interest in delivery applications in plant systems, we also assess the toxicity of polymer-based nanomaterials in plants and provide recommendations for future design of nanomaterial-based polynucleotide delivery strategies.

biochemistry↗

bioRxiv2021

Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves

Rapidly growing interest in nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influences transport in plants and use of their cargoes, limiting development and deployment of nanotechnology in plant systems. Here, we employ non-biolistically delivered DNA-modified gold nanoparticles (AuNP) spanning various sizes (5 - 20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana (Nb) leaves. Generally, smaller AuNPs demonstrate more rapid, higher, and longer-lasting levels of association with plant cell walls compared to larger AuNPs. We observe internalization of rod-shaped but not spherical AuNPs into plant cells, yet surprisingly, 10 nm spherical AuNP functionalized with small-interfering RNA (siRNA) are most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery, and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious bio-cargo delivery for future developments in plant nanobiotechnology.

bioengineering↗

bioRxiv2021

Gold nanocluster mediated delivery of siRNA to intact plant cells for efficient gene knockdown

RNA interference (RNAi), which involves the delivery of small interfering RNA molecules (siRNA), has been used to validate target genes in plants, to understand and control cellular metabolic pathways, and as a green alternative for crop pest tolerance. Conventional siRNA delivery methods such as viruses and Agrobacterium-mediated delivery exhibit limitations in host plant species range and their use can result in uncontrolled DNA integration into the plant host genome. Here, we synthesize polyethyleneimine functionalized gold nanoclusters (PEI-AuNCs) to mediate siRNA delivery into intact plant cells and show these constructs enable efficient gene knockdown. We demonstrate that functionalized AuNCs protect siRNA from RNase degradation and are small enough (~2 nm) to bypass the plant cell wall which exhibits a size exclusion limit of 5-20 nm. These AuNCs in turn enable up to 76.5 {+/-} 5.9% GFP mRNA knockdown efficiency with no cellular toxicity. Our data suggest this simple and biocompatible platform for passive delivery of siRNA into intact plant cells could have broad applications in plant biotechnology.

bioengineering↗

bioRxiv2019

Surface Modification Effects on Single-Walled Carbon Nanotubes for Multimodal Optical Applications

Optical nanoscale technologies often implement covalent or noncovalent strategies for the modification of nanoparticles, whereby both functionalizations are leveraged for multimodal applications but can affect the intrinsic fluorescence of nanoparticles. Specifically, single-walled carbon nanotubes (SWCNTs) can enable real-time imaging and cellular delivery; however, the introduction of covalent SWCNT sidewall functionalizations often attenuates SWCNT fluorescence. Herein, we leverage recent advances in SWCNT covalent functionalization chemistries that preserve the SWCNTs pristine graphitic lattice and intrinsic fluorescence and demonstrate that such covalently functionalized SWCNTs maintain fluorescence-based molecular recognition of neurotransmitter and protein analytes. We show that the covalently modified SWCNT nanosensor fluorescence response towards its analyte is preserved for certain nanosensors, presumably dependent on the steric hindrance introduced by the covalent functionalization that hinders noncovalent interactions with the SWCNT surface. We further demonstrate that these SWCNT nanosensors can be functionalized via their covalent handles to self-assemble on passivated microscopy slides, and discuss future use of these dual-functionalized SWCNT materials for multiplexed applications.

bioengineering↗

bioRxiv2019

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

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.

plant biology↗

bioRxiv2019

DNA Nanostructures Coordinate Gene Silencing in Mature Plants

Plant 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 constitutively-expressed 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.

plant biology↗