Getting Hold of the Tobamovirus Particle-Why and How? Purification Routes over Time and a New Customizable Approach.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of protein A (PA) on every coat protein (CP) subunit (TVCV) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCV and the wild-type subgroup 3 tobamovirus.

Bottom-Up Assembly of TMV-Based Nucleoprotein Architectures on Solid Supports.

RNA-guided self-assembly of tobacco mosaic virus (TMV)-like nucleoprotein nanotubes is possible using 3'-terminally surface-linked scaffold RNAs containing the viral origin of assembly (OAS). In combination with TMV coat protein (CP) preparations, these scaffold RNAs can direct the growth of selectively addressable multivalent carrier particles directly at sites of interest on demand. Serving as adapter templates for the installation of functional molecules, they may promote an integration of active units into miniaturized technical devices, or enable their presentation on soft-matter nanotube systems at high surface densities advantageous for, for example, biodetection or purification applications.

Dynamic DNA-controlled "stop-and-go" assembly of well-defined protein domains on RNA-scaffolded TMV-like nanotubes.

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.

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.

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).

The Impact of Aspect Ratio on the Biodistribution and Tumor Homing of Rigid Soft-Matter Nanorods.

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.

RNA-controlled assembly of tobacco mosaic virus-derived complex structures: from nanoboomerangs to tetrapods.

The in vitro assembly of artificial nanotubular nucleoprotein shapes based on tobacco mosaic virus-(TMV-)-derived building blocks yielded different spatial organizations of viral coat protein subunits on genetically engineered RNA molecules, containing two or multiple TMV origins of assembly (OAs). The growth of kinked nanoboomerangs as well as of branched multipods was determined by the encapsidated RNAs. A largely simultaneous initiation at two origins and subsequent bidirectional tube elongation could be visualized by transmission electron microscopy of intermediates and final products.

Tailoring the surface properties of tobacco mosaic virions by the integration of bacterially expressed mutant coat protein.

Due to its small dimensions and high stability, tobacco mosaic virus (TMV) is used as nano-scaffold frequently. Its surface can be engineered to meet specific needs for technical, medical or materials applications. However, not all technically desirable TMV coat protein (CP) mutants can be propagated in plants successfully, if they change the efficiency of virion assembly.

TMV nanorods with programmed longitudinal domains of differently addressable coat proteins.

The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly.

M1.3--a small scaffold for DNA origami .

The DNA origami method produces programmable nanoscale objects that form when one long scaffold strand hybridizes to numerous oligonucleotide staple strands. One scaffold strand is dominating the field: M13mp18, a bacteriophage-derived vector 7249 nucleotides in length. The full-length M13 is typically folded by using over 200 staple oligonucleotides.

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.

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.