Patterned Assembly of Genetically Modified Viral Nanotemplates via Nucleic Acid Hybridization (original) (raw)
Related papers
Langmuir, 2008
We demonstrate hierarchical assembly of tobacco mosaic virus (TMV)-based nanotemplates with hydrogel-based encoded microparticles via nucleic acid hybridization. TMV nanotemplates possess a highly defined structure and a genetically engineered high density thiol functionality. The encoded microparticles are produced in a high throughput microfluidic device via stop-flow lithography (SFL) and consist of spatially discrete regions containing encoded identity information, an internal control, and capture DNAs. For the hybridization-based assembly, partially disassembled TMVs were programmed with linker DNAs that contain sequences complementary to both the virus 5′ end and a selected capture DNA. Fluorescence microscopy, atomic force microscopy (AFM), and confocal microscopy results clearly indicate facile assembly of TMV nanotemplates onto microparticles with high spatial and sequence selectivity. We anticipate that our hybridization-based assembly strategy could be employed to create multifunctional viralsynthetic hybrid materials in a rapid and high-throughput manner. Additionally, we believe that these viral-synthetic hybrid microparticles may find broad applications in high capacity, multiplexed target sensing.
TMV Microarrays: Hybridization-Based Assembly of DNA-Programmed Viral Nanotemplates
Langmuir, 2007
The intrinsic ability of biological molecules to self-organize into complex structures has the potential to revolutionize methods for the assembly of nanomaterials and devices. In this work, nucleic acid hybridization was used to simultaneously assemble different Tobacco mosaic virus (TMV) nanotemplates onto a glass substrate patterned with address specific capture DNAs. To accomplish this, TMV-based nanotemplates were programmed with linker DNAs containing sequence specific addresses and hybridized directly to the capture DNAs. This assembly process proved to be a reliable, selective, and controllable means to assemble multiple TMV nanotemplates.
2012
The capability of some natural molecular building blocks to self-organize into defined supramolecular architectures is a versatile tool for nanotechnological applications. Their site-selective integration into a technical context, however, still poses a major challenge. RNA-directed self-assembly of tobacco mosaic virus-derived coat protein on immobilized RNA scaffolds presents a possibility to grow nucleoprotein nanotubes in place. Two new methods for their site-selective, bottom-up assembly are introduced. For this purpose, isothiocyanate alkoxysilane was used to activate oxidic surfaces for the covalent immobilization of DNA oligomers, which served as linkers for assembly-directing RNA. Patterned silanization of surfaces was achieved (1) on oxidic surfaces via dip-pen nanolithography and (2) on polymer surfaces (poly(dimethylsiloxane)) via selective oxidization by UV-light irradiation in air. Atomic force microscopy and X-ray photoelectron spectroscopy were used to characterize the surfaces. It is shown for the first time that the combination of the mentioned structuring methods and the isothiocyanate-based chemistry is appropriate (1) for the site-selective immobilization of nucleic acids and, thus, (2) for the formation of viral nanoparticles by bottom-up selfassembly after adding the corresponding coat proteins.
Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology
The self-assembly of viral building blocks bears exciting prospects for fabricating new types of bionanoparticles with multivalent protein shells. These enable a spatially controlled immobilization of functionalities at highest surface densities-an increasing demand worldwide for applications from vaccination to tissue engineering, biocatalysis, and sensing. Certain plant viruses hold particular promise because they are sustainably available, biodegradable, nonpathogenic for mammals, and amenable to in vitro self-organization of virus-like particles. This offers great opportunities for their redesign into novel "green" carrier systems by spatial and structural synthetic biology approaches, as worked out here for the robust nanotubular tobacco mosaic virus (TMV) as prime example. Natural TMV of 300 x 18 nm is built from more than 2,100 identical coat proteins (CPs) helically arranged around a 6,395 nucleotides ssRNA. In vitro, TMV-like particles (TLPs) may self-assemble also from modified CPs and RNAs if the latter contain an Origin of Assembly structure, which initiates a bidirectional encapsidation. By way of tailored RNA, the process can be reprogrammed to yield uncommon shapes such as branched nanoobjects. The nonsymmetric mechanism also proceeds on 3'terminally immobilized RNA and can integrate distinct CP types in blends or serially. Other emerging plant virus-deduced systems include the usually isometric cowpea chlorotic mottle virus (CCMV) with further strikingly altered structures up to "cherrybombs" with protruding nucleic acids. Cartoon strips and pictorial descriptions of major RNA-based strategies induct the reader into a rare field of nanoconstruction that can give rise to utile soft-matter architectures for complex tasks.
2020
Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them are the plant Tobacco mosaic virus (TMV) and Barley stripe mosaic virus (BSMV) due to their high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV, BSMV, and their virus-like particle (VLP) derivatives and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. We briefly outline how these features have been exploited in engineering applications such as sensing, catalysis, and energy storage, and discuss emerging advances that promise to accelerate the development of these biotemplates for widescale industrial use.
Plant virus directed fabrication of nanoscale materials and devices
Virology, 2015
Bottom-up self-assembly methods in which individual molecular components self-organize to form functional nanoscale patterns are of long-standing interest in the field of materials sciences. Such self-assembly processes are the hallmark of biology where complex macromolecules with defined functions assemble from smaller molecular components. In particular, plant virus-derived nanoparticles (PVNs) have drawn considerable attention for their unique self-assembly architectures and functionalities that can be harnessed to produce new materials for industrial and biomedical applications. In particular, PVNs provide simple systems to model and assemble nanoscale particles of uniform size and shape that can be modified through molecularly defined chemical and genetic alterations. Furthermore, PVNs bring the added potential to "farm" such bio-nanomaterials on an industrial scale, providing a renewable and environmentally sustainable means for the production of nano-materials. This...
Plant molecular farming of virus‐like nanoparticles as vaccines and reagents
WIREs Nanomedicine and Nanobiotechnology, 2019
The use of plants for the production of virus-like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus-derived vectors for the transient expression of engineered viral or other structural proteins in plants-a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience-whole plants are generally used, and processes do not need to be sterile-and cost, as bulk biomass production is significantly cheaper than by any other method. Plant-made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP-forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi-and rotavirus particles that require assembly of four co-expressed proteins, and polio-and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4-component VNPs. Both plant and animal virus-derived plant-made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging
Journal of General Virology, 2012
An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein-RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.
ACS applied materials & interfaces, 2016
Using tobacco mosaic virus coat proteins (TMVcp) from both sources of the plant and bacterial expression systems as building blocks, we demonstrate here a coassembly strategy of TMV nanotubes in the presence of RNA. Specifically, plant-expressed cp (cpp) efficiently dominates the genomic RNA encapsidation to determine the length of assembled TMV nanotubes, whereas the incorporated Escherichia coli-expressed cp (cpec) improves the physical stability of TMV nanotubes by introducing disulfide bonds between the interfaces of subunits. We expect this coassembly strategy can be expanded to other virus nanomaterials to obtain desired properties based on rationally designed protein-RNA and protein-protein interfacial interactions.