Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies.

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'.

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes.

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.

Interaction of human plasma proteins with thin gelatin-based hydrogel films: a QCM-D and ToF-SIMS study.

In the fields of surgery and regenerative medicine, it is crucial to understand the interactions of proteins with the biomaterials used as implants. Protein adsorption directly influences cell-material interactions in vivo and, as a result, regulates, for example, cell adhesion on the surface of the implant. Therefore, the development of suitable analytical techniques together with well-defined model systems allowing for the detection, characterization, and quantification of protein adsorbates is essential. In this study, a protocol for the deposition of highly stable, thin gelatin-based films on various substrates has been developed. The hydrogel films were characterized morphologically and chemically. Due to the obtained low thickness of the hydrogel layer, this setup allowed for a quantitative study on the interaction of human proteins (albumin and fibrinogen) with the hydrogel by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). This technique enables the determination of adsorbant mass and changes in the shear modulus of the hydrogel layer upon adsorption of human proteins. Furthermore, Secondary Ion Mass Spectrometry and principal component analysis was applied to monitor the changed composition of the topmost adsorbate layer. This approach opens interesting perspectives for a sensitive screening of viscoelastic biomaterials that could be used for regenerative medicine.

New approaches for bottom-up assembly of tobacco mosaic virus-derived nucleoprotein tubes on defined patterns on silica- and polymer-based substrates.

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 self-assembly after adding the corresponding coat proteins.

Inducible site-selective bottom-up assembly of virus-derived nanotube arrays on RNA-equipped wafers.

Tobacco mosaic virus (TMV) is a tube-shaped, exceptionally stable plant virus, which is among the biomolecule complexes offering most promising perspectives for nanotechnology applications. Every viral nanotube self-assembles from a single RNA strand and numerous identical coat protein (CP) subunits. Here we demonstrate that biotechnologically engineered RNA species containing the TMV origin of assembly can be selectively attached to solid surfaces via one end and govern the bottom-up growth of surface-linked TMV-like nanotubes in situ on demand. SiO(2) wafers patterned by polymer blend lithography were modified in a chemically selective manner, which allowed positioning of in vitro produced RNA scaffolds into predefined patches on the 100-500 nm scale. The RNA operated as guiding strands for the self-assembly of spatially ordered nanotube 3D arrays on the micrometer scale. This novel approach may promote technically applicable production routes toward a controlled integration of multivalent biotemplates into miniaturized devices to functionalize poorly accessible components prior to use. Furthermore, the results mark a milestone in the experimental verification of viral nucleoprotein complex self-assembly mechanisms.