The robust, anisotropic tobacco mosaic virus (TMV) provides a monodisperse particle size and defined surface chemistry. Owing to these properties, it became an excellent bio-template for the synthesis of diverse nanostructured organic/inorganic functional materials. For selective mineralization of the bio-template, specific functional groups were introduced by means of different genetically encoded amino acids or peptide sequences into the polar virus surface. An alternative approach for TMV surface functionalization is chemical coupling of organic molecules. To achieve mineralization control in this work, we developed a synthetic strategy to manipulate the surface hydrophilicity of the virus through covalent coupling of polymer molecules. Three different types of polymers, namely the perfluorinated (poly(pentafluorostyrene) (PFS)), the thermo-responsive poly(propylene glycol) acrylate (PPGA), and the block-copolymer polyethylene-block-poly(ethylene glycol) were examined. We have demonstrated that covalent attachment of hydrophobic polymer molecules with proper features retains the integrity of the virus structure. In addition, it was found that the degree of the virus hydrophobicity, examined via a ZnS mineralization test, could be tuned by the polymer properties.

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g. peptides and synthetic molecules. Multiple modifications enable uses including vaccination, biosensing, tissue engineering, intravital delivery and theranostics. Novel concepts exploit self-organization capacities of viral building blocks into hierarchical 2D and 3D structures, and their conversion into biocompatible, biodegradable units. High yields of VNPs and proteins can be harvested from plants after a few days so that various products have reached or are close to commercialization. The article delineates potentials and limitations of biomedical plant VNP uses, integrating perspectives of chemistry, biomaterials sciences, molecular plant virology and process engineering.

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g. peptides and synthetic molecules. Multiple modifications enable uses including vaccination, biosensing, tissue engineering, intravital delivery and theranostics. Novel concepts exploit self-organization capacities of viral building blocks into hierarchical 2D and 3D structures, and their conversion into biocompatible, biodegradable units. High yields of VNPs and proteins can be harvested from plants after a few days so that various products have reached or are close to commercialization. The article delineates potentials and limitations of biomedical plant VNP uses, integrating perspectives of chemistry, biomaterials sciences, molecular plant virology and process engineering. (C) 2018 Elsevier B.V. All rights reserved.

Bioinspired mineralization for the production of new functional materials offers mild reaction conditions suitable for integrating biological templates and build hierarchically organized hybrid nanostructures with defined properties. In this respect, the tobacco mosaic virus (TMV) stands out due to its unique structural dimensions. Here, the authors present a novel mineralization pathway for the synthesis of virus-based zinc oxide (ZnO) hybrids with multifunctional properties. Wild-type TMV, two TMV mutants (E50Q and TMV-Cys) and amino-functionalized self-assembled monolayers (NH2-SAMs), as a reference, were used as templates. This mineralization approach allows control of the particle size of the inorganic phase. Further, the virus contributes additionally to the texturing of zinc oxide. Field-effect transistors (FETs) built from the hybrid films, obtained at close to ambient conditions, showed reproducible results at optimized conditions and without posttreatment. This significantly reduced the threshold voltage of the E50Q/zinc oxide FET compared to that of the NH2-SAMs/zinc oxide FET points to the impact of the organic template on FET performance. Nacre-like virus-based zinc oxide multilayers and corresponding monolithic references were prepared. The mechanical properties, namely, Young's modulus, hardness and fracture toughness, were determined and an improvement in the mechanical performance by genetic modification was observed.

Taking advantage of the ability for in vitro assembly of the plant-infecting virus tobacco mosaic virus (TMV), rod-shaped nanoscale scaffolds presenting different addressable groups can be obtained. We have established procedures resulting in virus-like particles with randomly distributed functional groups, with different groups arranged in striped but randomized structures, and even with distinct groups clustered in adjacent, better-defined domains. The TMV coat protein (CP) variants combined in these approaches can either originate all from TMV mutants propagated in planta, or be mixed with CP expressed in E. coli (CPEc). Protocols for expression and purification of a CPEc-His6 mutant in E. coli as well as the different methods for in vitro assembly and the visualization by decoration of one CP type are explained in detail.

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (M-n = 700 g mol(-1)) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

There is an increasing interest in the application of Michael addition reactions, which makes it possible to obtain simply and highly effective new materials at environmentally friendly conditions. This study employed this method to obtain tobacco mosaic virus (TMV)-based hybrid materials as interesting precursors for different applications. Therefore, the authors investigated the linker-enabled coupling of a cysteine-exposing mutant TMVCys nanorod that displays a thiol group on each of its 2130 protein subunits. These can undergo a thiol-Michael reaction with corresponding functional groups of different linkers to form several types of coupling products. The linkers varied in their central aryl or heteroaryl unit, the spacer length with different amounts of CH2 groups and the respective functional groups such as methacrylamide, acrylamide and maleimide. It could be shown that not only the reactivity of the Michael acceptor plays an important role in product formation but also the linker length and the composition of the core group.

The bioinspired synthesis of hierarchical hybrid nanomaterials using biological objects as a template attracts growing interest for the design of new technologically relevant nanostructured materials. To ensure control over the shape and properties of the fabricated hybrid structures, understanding of the growth mechanism is required. In this work, tobacco mosaic virus (TMV) is used as a template to direct the synthesis of zinc sulfide (ZnS) at ambient conditions and different pH from additive-free aqueous solution. TMV/ ZnS hybrid nanowires or thin films are obtained with controllable thickness of the inorganic layer. The deposition mechanism is studied by monitoring the optical properties, band gap (E-g) and particle size, respectively, of ZnS particles mineralized on the TMV template and ZnS reference nanoparticles. A heterogeneous nucleation of the inorganic phase on the template surface is proposed. Band gap measurements reveal that the average size of the ZnS nanoparticles grown on the virus surface is smaller compared to solution-grown nanoparticles. Moreover, a blue shift of the ZnS photoluminescence peak indicates a dominance of different crystal lattice defects in both systems. The present method for the selective template-directed mineralization opens new possibilities in the synthesis of well-organized functional hybrid materials.

A new approach for the development of electrochemical biosensors using tobacco mosaic virus (TMV) nanotubes and coat protein (CP) aggregates as enzyme nanocarriers is presented. The generic approach has been demonstrated by realizing amperometric glucose sensors combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes and CPs as a model system. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of enzymes without substantial loss of their activity. The obtained results underline the potential of integrating biomolecule/virus hybrids with electronic transducers for applications in biosensorics.

PmrA, an OmpR/PhoB family response regulator (RR), manages genes for antibiotic resistance. Phosphorylation of OmpR/PhoB RR induces the formation of a symmetric dimer in the N-terminal receive domain (REC), promoting 2 C-terminal DNA-binding domains (DBDs) to recognize the promoter DNA to elicit adaptive responses. Recently, determination of the KdpE-DNA complex structure revealed a unique asymmetric REC-DBD interface that may be necessary for transcription activation. Here, we report the 3.2-Å resolution of the crystal structure of the PmrA-DNA complex, which reveals a similar yet different asymmetric REC-DBD interaction. However, this asymmetric interface is transiently populated in solution, as revealed by relaxation dispersion NMR, and is not crucial in supporting gene expression, as evidenced by the b-galactosidase reporter assay. The interdomain dynamics of the protein, together with the DBD-DBD interface, may help PmrA search for the most suitable conformation for interacting with RNA polymerase holoenzyme to activate downstream gene transcription.

Biomineralization in general is based on electrostatic interactions and molecular recognition of organic and inorganic phases. These principles of biomineralization have also been utilized and transferred to bio-inspired synthesis of functional materials during the past decades. Proteins involved in both, biomineralization and bio-inspired processes, are often piezoelectric due to their dipolar character hinting to the impact of a template's piezoelectricity on mineralization processes. However, the piezoelectric contribution on the mineralization process and especially the interaction of organic and inorganic phases is hardly considered so far. We herein report the successful use of the intrinsic piezoelectric properties of tobacco mosaic virus (TMV) to synthesize piezoelectric ZnO. Such films show a two-fold increase of the piezoelectric coefficient up to 7.2 pm V-1 compared to films synthesized on non-piezoelectric templates. By utilizing the intrinsic piezoelectricity of a biotemplate, we thus established a novel synthesis pathway towards functional materials, which sheds light on the whole field of biomimetics. The obtained results are of even broader and general interest since they are providing a new, more comprehensive insight into the mechanisms involved into biomineralization in living nature.

Virus particles and proteins are excellent examples of naturally occurring structures with well-defined nanoscale architectures, for example, cages and tubes. These structures can be employed in a bottom-up assembly strategy to fabricate repetitive patterns of hybrid organic–inorganic materials. In this paper, we review methods of assembly that make use of protein and virus scaffolds to fabricate patterned nanostructures with very high spatial control. We chose (apo)ferritin and tobacco mosaic virus (TMV) as model examples that have already been applied successfully in nanobiotechnology. Their interior space and their exterior surfaces can be mineralized with inorganic layers or nanoparticles. Furthermore, their native assembly abilities can be exploited to generate periodic architectures for integration in electrical and magnetic devices. We introduce the state of the art and describe recent advances in biomineralization techniques, patterning and device production with (apo)ferritin and TMV.

The rod-shaped nanoparticles of the widespread plant pathogen tobacco mosaic virus (TMV) have been a matter of intense debates and cutting-edge research for more than a hundred years. During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus-host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

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 (cp(p)) efficiently dominates the genomic RNA encapsidation to determine the length of assembled TMV nanotubes, whereas the incorporated Escherichia coli-expressed cp (cp(ec)) 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.

The conjunction of (bio-)chemical recognition elements with nanoscale biological building blocks such as virus particles is considered as a very promising strategy for the creation of biohybrids opening novel opportunities for label-free biosensing. This work presents a new approach for the development of biosensors using tobacco mosaic virus (TMV) nanotubes or coat proteins (CPs) as enzyme nanocarriers. Sensor chips combining an array of Pt electrodes loaded with glucose oxidase (GOD)-modified TMV nanotubes or CP aggregates were used for amperometric detection of glucose as a model system for the first time. The presence of TMV nanotubes or CPs on the sensor surface allows binding of a high amount of precisely positioned enzymes without substantial loss of their activity, and may also ensure accessibility of their active centers for analyte molecules. Specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CPs was achieved via bioaffinity binding. These layouts were tested in parallel with glucose sensors with adsorptively immobilized [SA]-GOD, as well as [SA]-GOD crosslinked with glutardialdehyde, and came out to exhibit superior sensor performance. The achieved results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for future applications in biosensorics and biochips. (C) 2016 Elsevier B.V. All rights reserved.

A DNA-based approach allows external control over the self-assembly process of tobacco mosaic virus (TMV)-like ribonucleoprotein nanotubes: their growth from viral coat protein (CP) subunits on five distinct RNA scaffolds containing the TMV origin of assembly (OAs) could be temporarily blocked by a stopper DNA oligomer hybridized downstream (3') of the OAs. At two upstream (5') sites tested, simple hybridization was not sufficient for stable stalling, which correlates with previous findings on a non-symmetric assembly of TMV. The growth of DNA-arrested particles could be restarted efficiently by displacement of the stopper via its toehold by using a release DNA oligomer, even after storage for twelve days. This novel strategy for growing proteinaceous tubes under tight kinetic and spatial control combines RNA guidance and its site-specific but reversible interruption by DNA blocking elements. As three of the RNA scaffolds contained long heterologous non-TMV sequence portions that included the stopping sites, this method is applicable to all RNAs amenable to TMV CP encapsidation, albeit with variable efficiency most likely depending on the scaffolds' secondary structures. The use of two distinct, selectively addressable CP variants during the serial assembly stages finally enabled an externally configured fabrication of nanotubes with highly defined subdomains. The "stop-and-go" strategy thus might pave the way towards production routines of TMV-like particles with variable aspect ratios from a single RNA scaffold, and of nanotubes with two or even more adjacent protein domains of tightly pre-defined lengths.

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50 % of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional 'high-binding' microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

The size and shape of nanocarriers can affect their fate in vivo, but little is known about the effect of nanocarrier aspect ratio on biodistribution in the setting of cancer imaging and drug delivery. The production of nanoscale anisotropic materials is a technical challenge. A unique biotemplating approach based on of rod-shaped nucleoprotein nanoparticles with predetermined aspect ratios (AR 3.5, 7, and 16.5) is used. These rigid, soft-matter nanoassemblies are derived from tobacco mosaic virus (TMV) components. The role of nanoparticle aspect ratio is investigated, while keeping the surface chemistries constant, using either PEGylated stealth nanoparticles or receptor-targeted RGD-displaying formulations. Aspect ratio has a profound impact on the behavior of the nanoparticles in vivo and in vitro. PEGylated nanorods with the lowest aspect ratio (AR 3.5) achieve the most efficient passive tumor-homing behavior because they can diffuse most easily, whereas RGD-labeled particles with a medium aspect ratio (AR 7) are more efficient at tumor targeting because this requires a balance between infusibility and ligand-receptor interactions. The in vivo behavior of nanoparticles can therefore be tailored to control biodistribution, longevity, and tumor penetration by modulating a single parameter: the aspect ratio of the nanocarrier.