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WIREs Nanomed Nanobiotechnol
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From stars to stripes: RNA‐directed shaping of plant viral protein templates—structural synthetic virology for smart biohybrid nanostructures

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Abstract 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. This article is categorized under: Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Biology‐Inspired Nanomaterials > Nucleic Acid‐Based Structures
Conceptual interrelation between the major subject of this article and optional research area definitions: suggested straightened use of terms. Left: Structurally defined nanocarrier scaffolds, for example, of viral origin, with selectively addressable subdomains (bright/dark blue) may be equipped with groups of functional ensembles of collaborating (bio) molecules (yellowish “modules”). Right: While the spacing and orientation of the individual collaborating partner molecules within modules is a matter of “spatial synthetic biology” (yellow) according to Polka et al., , the combined functional unit of modules and nanocarrier scaffolds serving as immobilization platform may be called a higher‐order subject of “structural synthetic biology” (green). It includes both the spatial arrangement within modules and their ordered display on immobilization scaffolds, slightly expanding the initial definition of Chen et al., . The generation of artificial, functional designer assemblies from (plant) viral building blocks is now called “synthetic (plant) virology” (Chen, Butler, Chen, & Suh, ; Saunders & Lomonossoff, ; Steele et al., ), with multiple design options and resulting application perspectives for synthetic, virus‐based scaffolds
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Altering the self‐assembly of a spherical ssRNA virus to form tubular nanostructures with dsDNA. (a) Under the conditions applied (see text), assembly of CCMV CPs has to be supported by a scaffold, due to weak protein–protein interactions. CCMV CP dimers were assembled in the presence of a nonviral, nonspecific dsDNA (500 bp) in distinct stoichiometric ratios of (A) 1:1, (B) 7:1, (C) 14:1 and (D) 28:1 bp:CP dimer. Ratios up to 10:1 bp:CP dimers resulted in tubes with uniform 17 nm diameter, but varying length. At ratios > > 10:1 bp:CP dimer (as in D), large structures with increased diameter up to 100 nm were found, as based on DNA condensates. Samples were negatively stained and analyzed by TEM. Reprinted with permission from Mukherjee, Pfeifer, Johnson, Liu, and Zlotnick (). Copyright (2006) American Chemical Society. (b) Schematic illustration of the CP dimer‐DNA assembly. Tubes longer than 170 nm are probably formed by a scaffold of staggered DNA strands. Starting from a nucleation center, several strands of DNA and CP can be recruited to elongate the tube as long as both partners are present. Adapted with permission from Mukherjee et al. (). Copyright (2006) American Chemical Society. (c) Assembled rods as in (a) were treated with different pH values by mixing with citrate buffer to examine the effect of pH onto the overall structure. Samples were negatively stained and examined by TEM. pH 7.5: assembled tubes; pH 6.0: fresh dilution of CP dimer assembled into 28 nm empty particles; pH 5.5: rod structure is remolded, small fractions are blebbing off the ends of the rods; pH 4.5: further destruction of the tube structure, few 28 nm particles are visible; pH 3.5: tube structure is completely deformed, particles are distorted. Reprinted with permission from Burns, Mukherjee, Keef, Johnson, and Zlotnick (). Copyright (2010) American Chemical Society. CCMV, cowpea chlorotic mottle virus; CP, coat protein; TEM, transmission electron microscopy
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Formation of supershapes by self‐assembly of CPs from cowpea chlorotic mottle virus (CCMV) with RNA molecules of increasing length, or RNA/DNA hybrids. (a) Scheme of the encapsidation of RNA of “allowed” length (1), and “overlong” RNA of roughly twice the normal size (2). (b) Negative‐stain TEM images of the assembly products. Structure types observed for varying RNA lengths: singlets (3 kb), doublets (6 kb), triplets (9 kb) and quadruplets (12 kb). (c) Cartoon depicting the RNA‐guided assembly of CCMV CPs to “cherrybombs” and “raspberries”. (1) Incubation of a 3 kb‐ssRNA with a complementary ssDNA strand. (2) Hybridization of RNA and DNA forms a hybrid scaffold. (3) Addition of CCMV CPs to the RNA–DNA template initiates capsid formation. (4) Capsid formation is completed. The major RNA portion is selectively encapsidated, but the RNA–DNA hybrid is poking out of the capsid through a small hole (“cherry‐bomb” structure). Image b is reprinted with permission from Cadena‐Nava et al. () and image d is reprinted with permission from Garmann et al. (). Copyright (2015) American Chemical Society). CP, coat protein
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Assembly of magnetic biocomposite materials by fusion of TMV and magnetosomes. (a) Schematic representation of the precise assembly process of functionalized magnetosomes and modified TMV scaffolds. (1) Magnetosomes are genetically designed to display green fluorescent binding nanobody proteins or streptavidin in their membrane (black bows). (2) TMV scaffolds are densely decorated with GFP or biotin (green) over the whole surface. (3) Mixing of TMV and magnetosomes, both functionalized with the complementary recognition sites, leads to the formation of complex strand‐like aggregates, magnetosomes hereby completely covering the underlying multiple‐particle TMV scaffold. (4) TMV particles are specifically functionalized with a few GFP or biotin molecules on their 5′‐protein end. (5) Mixing of single end‐modified TMV particles with complementary magnetosomes forms star‐like or pearl‐necklace‐like structures with magnetosomes at only one end of the TMV rods. (b) TEM image of complex strand‐like aggregates found after incubation of magnetosomes with entirely functionalized TMV particles (as depicted in (3)). (c) TEM image of a pearl‐necklace‐like structure found in mixtures with 5′‐terminally functionalized TMV particles (schematically illustrated in (5)). Images b and c are reprinted with permission from Mickoleit et al. (). Copyright (2018) American Chemical Society. TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Formation of fine‐tuned superlattice wires based on TMV and inorganic nanoparticles. (a) Scheme demonstrating the cooperative self‐assembly process of protein‐metal wires from TMV nanorods and spherical gold‐nanoparticles (AuNP). (1) Nucleation occurs between TMV particles (blue) and gold‐dots (yellow). (2) Individual AuNPs interact with viral rods. (3) Two or more TMV particles are crosslinked by numerous AuNPs. (4) Formation of a well‐ordered TMV‐AuNP superlattice. (b) TEM images of progressive assembly‐steps with different particle stoichiometries, as depicted in (a). Reproduced according to the Creative Commons Attribution 4.0 International Public License from Liljeström et al. (). TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Bottom‐up growth of TMV‐like structures on gold‐nanoparticles to build nanostars. (a) Cartoon strip illustrating the bottom‐up assembly of nanostars with gold cores (AuNPs) and nanotube‐arms derived from TMV. (1) AuNPs are functionalized through thiol–Au bonds with short oligonucleotide sequences (blue), which are reverse complementary to the 3′‐end of the TMV‐based RNA. (2) RNA constructs (black), carrying the OAs, are hybridized to the DNA coating. (3) Immobilized RNA is encapsidated by TMV CPs forming numerous straight arms of TMV‐like nanotubes. (b) TEM image showing star‐shaped architectures with gold core and straight TMV‐like nanorods as arms. Reprinted with permission from Eber, Eiben, Jeske, and Wege (). CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Self‐organization of engineered TMV CP (mutant T103C) nanorings into gear‐wheel like superstructures under alkaline conditions. (a) Scheme of three additional mutations responsible for the superior disk stability under alkaline conditions (53R) and the shape switching behaviour (in combination of T103C, 53R, 1 K and 158 K mutations) of multimeric disk assemblies. (2) Layout of a “gear‐wheel” composed of several (here: 12) disks each interacting with two adjacent ones at a single attachment site only. (b) TEM image of such assemblies formed at pH 9.5. Image b is reprinted with permission from Zhang et al. (). CP, coat protein; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Fabrication of planar TMV‐based crystals not dictated by RNA. (a) Cartoon illustrating the crystallization process of different TMV CP disk variants stabilized by internal disulphide bonding between individual CP subunits (mutant T103C) and, additionally, (1) exposing either cysteine (Cys, yellow) or histidine (His) residues (dark green) on each CP. (2) Suitable conditions enable a controlled crystallization process. Cysteine mutant: Low temperature (4°C) in combination with a slightly acidic pH (pH 5) supports direct bridging of discrete CPs via disulfide‐bond formation of the Cys thiols within one month. Histidine mutant: Crystal formation is carried out by incubation of His‐exposing CPs and Zn2+‐ions at room temperature, in this metal−His chelation, Zn2+ acts as a cross‐linker. (b) TEM image of Cys‐exposing TMV CP assembled via disulfide‐bonds into triclinic crystals. (c) TEM image of TMV CP‐His containing crystals in high magnification. The strong metal‐His chelation resulted in closely packed hexagonal TMV assemblies. Images b and c are reprinted with permission from Zhang, Wang, et al. (). Copyright (2018) American Chemical Society. CP, coat protein; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Assembly of fluorescently labeled and DNA‐programmed TMV‐based nanotemplates onto hydrogel microparticles. (a) Scheme depicting the partial disassembly of cysteine‐presenting TMV variants (TMVCys) to expose accessible RNA on the 5′‐end and coupling of fluorescein‐maleimide to cysteine thiol groups. Labeled and stripped TLPs are “programmed” via hybridization with DNA adapter oligonucleotides consisting of an RNA‐complementary region (green) and a region complementary (blue) to the capture DNA sequences (orange) embedded in PEG‐based microparticles. (b) Schematic diagram showing the synthesis of the PEG microparticles via stop‐flow lithography in a high throughput microfluidic device. Microparticles consist of three regions: an encoded part containing Rhodamine B (red), a control middle section (white) and a segment with capture DNAs embedded (green). Programmed TMV nanotemplates are hybridized with the DNA on the microparticles. (c) Cartoon illustrating a magnified area with TMV hybridization onto the PEG‐polymer particles (gray network). DNA linker oligonucleotides hybridized to TMV RNA (blue/green) for programming bind to the reverse‐complementary capture DNA sequences (orange). (d) Brightfield microscopy image of the 180 μm × 90 μm × 30 μm microparticles. (e) Fluorescence overlay image of microparticles (as in b): fluorescein‐labeled TMVCys assemblies after hybridization (green), Rhodamine B (red), control region (black). Images b, d and e are adapted with permission from Tan et al., ). Copyright (2008) American Chemical Society. PEG, polyethylene glycol; TMV, tobacco mosaic virus
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Fabrication of TMV microarrays based on nucleic acid hybridization. (a) Cartoon illustrating the hybridization‐based programming of microarray platforms with TMV nanotemplates. (1) Functional thiol groups‐exposing variants of TMV (TMVCys, light blue) are labeled either with the fluorescent dyes Cy5 (red) or Cy3 (green), and partially disassembled to expose small 5′‐portions of the viral RNA. (2) Partially disassembled TMVs are hybridized with specific DNA adapter oligonucleotides (green or red). (3) Equal mixtures of Cy5‐ and Cy3‐labeled TMV‐templates are incubated on glass substrates patterned with complementary capture DNAs for hybridization. After washing and drying, microarrays are scanned for fluorescence. (b) Fluorescence scan of labeled and programmed TMV structures hybridized onto microarrays. Reprinted with permission from Yi et al. (). Copyright (2007) American Chemical Society. TMV, tobacco mosaic virus
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Fabrication of expanded tetrahedral nucleoprotein structures based on organic DNA‐hybrid branching elements, RNA scaffolds and TMV coat protein. (a) Schematic representation of a novel TMV‐functionalized nanostructure (with just a single arm shown for clarity). A tetrahedral adamantane‐based organic core with four short DNA arms (DNA‐hybrid, gray) is chemically linked to a longer DNA‐linker sequence (blue). These extended DNA‐hybrids are enzymatically phosphorylated and ligated to short RNA molecules containing the TMV OAs (black), followed by encapsidation of the RNA between TMV CPs and thus tube formation (light blue) in situ. Alternatively, prefabricated TMV tubes (light blue) with short 3′‐portions of accessible RNA (black, held free by way of DNA blocking elements not depicted here) are ligated with the branched DNA‐terminated cores. Linkage of nucleoprotein structures can occur on one to four hybrid‐arms resulting in up to four‐armed 3D nanoobjects, shown in (b). (b) Representative TEM images of long prefabricated TMV rods either in reaction mixtures devoid of T4 RNA ligase 2 serving as negative control (1), or linked to two (2), three (3) or all four arms (4) of the branched DNA‐hybrid core. Particles were visualized using small‐angle Pt shadowing. Reproduced from Wenz et al. () with permission from The Royal Society of Chemistry. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Fabrication of kinked nanostructures scaffolded by designed RNA molecules and TMV CP. (a) Scheme describing the proposed assembly model for kinked and branched TMV nanotube constructs. (1) Artificial RNA scaffolds carrying two or more tandemly repeated OAs sequences (black) are mixed with TMV CPs and disks (blue). (2) Nanotube growth nucleates almost simultaneously at each OAs segment and is continued via further disks and CPs. (3) Growing TMV nanorod arms collide (in a collision zone) and stop in growth, resulting in a final nanoboomerang shape or higher‐order‐kinked multipods. (b) TEM images illustrating the progressive nanotube formation occurring simultaneously on one RNA molecule with two or more OAs sites, thereby forming kinked nanoboomerangs as well as branched tri‐ and tetrapods. Samples were spread and rotary‐shadowed to visualize free RNA (1) or negatively stained with uranyl acetate (2). Reproduced [except for bottom image in (1) that was taken in parallel] from Eber et al. () with permission from The Royal Society of Chemistry. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Formation of virus‐like particles with randomly distributed CP species exposing different functional groups. (a) Mixtures of different CP types (light and dark blue) and RNA molecules containing the TMV OAs (black) assemble into stochastically mixed rods. (b) Native agarose gel revealing the composition of mixed particle types with gradually increased CPLys fraction, as indicated above. Lanes (1) and (2) show TMVWT and TMVLys particles isolated from tobacco, respectively. Reprinted from Eiben et al. () with permission from Springer
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Fabrication of two or three distinct longitudinal domains of different coat proteins (CPs) on a single virus‐like particle. (a) Cartoon strip illustrating a two‐step in vitro assembly process of viral or designed RNA molecules (black), containing the TMV OAs, and CP‐mutants carrying either amino (from TMVLys) or thiol (from TMVCys) groups on their outer surface. (1) RNA and limited amounts of a first CP species (light blue) are mixed under suitable assembly conditions. (2) The assembly process starting at the OAs sequence results in partly assembled nanorods with freely accessible 3′‐ and 5′‐portions of the RNA. (3) Addition of a second CP species (dark blue) leads to fully assembled particles of 300 nm length consisting of adjacent longitudinal domains of differently addressable CP species. The cysteine‐exposing domains (light blue, CP1) are made visible by coupling of maleimide‐PEG‐biotin linkers (linker) and subsequent decoration with streptavidin–horseradish peroxidase conjugates (gray clouds). (b) TEM image of TMV‐like particles with neighboring domains of two CP species and enzyme decoration as in (a3). Reproduced from Geiger et al. () with permission from The Royal Society of Chemistry. (c) Schematic of the “stop‐and‐go” assembly process to fabricate two well‐defined protein domains on an RNA‐scaffolded TMV particle by help of dynamic DNA nanotechnology. (1) A short stopper‐DNA oligonucleotide (blue) is partially hybridized downstream (3') the OAs sequence of a viral RNA molecule, with an overhang (“toehold”) protruding. (2) A first CP species (light blue) is added leading to partial particle formation. Stalled TMV‐like nanorods are purified from unassembled CPs via ultracentrifugation. (3) Addition of a “fuel” DNA oligomer enabling toehold‐mediated release (yellow, fully complementary to the stopper oligomer) removes the stopper from the RNA. Upon use of a second CP species (dark blue), particle growth is taken up again and completed. Lysine‐exposing CPs (dark blue, CP2) are functionalized with NHS‐PEG‐biotin linkers (linker) and streptavidin–horseradish peroxidase conjugates (gray clouds). (d) TEM image of TMV‐like nanotubes with two subdomains of distinct CP variants and enzyme decoration as in (c3). Reproduced from Schneider et al. () with permission from The Royal Society of Chemistry. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Bottom‐up growth of TMV‐like particles on elongated DNA origami. (a) Schematic model illustrating the in situ self‐assembly of TMV‐like structures on rigid 24HB DNA origami (gray). Two 40 nt long capture strands (blue) allow site specific anchoring of the 3′‐end of designed RNAs containing the TMV OAs (black). Addition of viral CPs initiates the encapsidation process. (b) TEM images of assembled TMV nanorods with different length (corresponding to varying RNA length) grown on DNA origami scaffolds. Scale bars: 50 nm. Adapted with permission from Zhou, Ke, and Wang (). Copyright (2018) American Chemical Society. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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Scheme of the assembly stages of TMV nanoparticles from OAs‐containing RNA and CP subunits. Refer to the text for a thorough description. (a) Shows the successive assembly intermediates and the overall movement of the RNA scaffold upon its encapsidation. (b) Visualizes the organization of the final particle and its representation as applied in subsequent figures, with the RNA helix sandwiched between the helically arranged CP subunits. Images are not to scale. Adapted according to the Creative Commons Attribution 2.0 International Public License from Koch et al. (). CP, coat protein; OAs, origin of assembly; TMV, tobacco mosaic virus
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Bottom‐up growth of viral tubes linked to solid surfaces via DNA adapters. (a) Schematic of RNA‐guided bottom‐up assembly of TMV nanotubes covalently linked to solid surfaces. Amino‐terminated DNA linker molecules are immobilized on those surfaces by application of aldehyde surface functionalization and Schiff base condensation. Free 5′‐ends end of the DNA linkers serve as RNA anchors; they are linked enzymatically to RNA containing the TMV OAs. Addition of purified TMV CPs under conditions driving bottom‐up self‐assembly leads to the formation of terminally immobilized virus‐like nanorods. (b) AFM topography images of a SiO2 wafer patterned by polymer blend lithography with aldehyde‐exposing areas and utilized for bottom‐up assembly of TMV nanorods. The magnified area (1 μm2) is marked with a white/black frame. Images reprinted with permission from Mueller et al. (). (c) AFM topography image of a silicon wafer substrate fashioned with TMV‐like nanotubes grown on hybridization‐linked RNA. Here, bottom‐up self‐assembly was achieved after isothiocyanate (ITC) silane‐based surface functionalization, covalent coupling of amino‐terminated DNA linkers through thiourea bond formation, and RNA binding by hybridization, followed by CP input. Scan area: 3 μm x 3 μm. Reproduced with permission from Azucena et al. (). Copyright (2012) American Chemical Society. CP, coat protein; OAs, origin of assembly; TMV, tobacco mosaic virus
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Design of viral nanostructures with altered length and thus aspect ratio. (a) Fabrication of exceptionally short RNA‐assisted virus‐like particles. TMV CPs and short RNA‐scaffolds containing the OAs sequence form ring‐shaped four‐turn helices (“disks”). (b) TEM analysis of nucleoprotein particles generated from the short RNAs and viral CP, as in (a). Brackets indicate four‐turn helices. Reproduced with permission from Altintoprak et al. (). (c) Schematics of TMV particles in various lengths. (d) TEM image of mixed length‐modified virus particles, as in (c), with variable aspect ratio. CP, coat protein; OAs, origin of assembly; TEM, transmission electron microscopy; TMV, tobacco mosaic virus
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