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. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CP(Cys) and CP(Lys) with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.

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.

Current models for magnetoviscosity suggest that replacing the spherical nanoparticles of a conventional ferrofluid with magnetic nanotubes would lead to a stronger field‐induced viscosity enhancement and a much‐improved stability against shear thinning – two important parameters for technological exploitation of the magnetoviscous effect. We report the development of positive and negative templating strategies for the synthesis of magnetic nanotubes out of a variety of materials. Our positive template is Tobacco mosaic virus (TMV) – in natural form or genetically engineered to express specific surface chemistries and lengths – which we exploit as a template for the electroless deposition (ELD) of nanosized clusters of nickel and as a scaffold for magnetic particles in a conventional ferrofluid. Our negative templating strategy employs porous anodic aluminum oxide (AAO) as a substrate for the atomic layer deposition (ALD) of a conformal coating of iron oxide, offering precise control over the length and wall thickness of the resulting nanotubes. Both strategies were scaled up to produce the mass quantities of uniform‐aspect‐ratio nanotubes that are needed for macroscopic ferrofluid volumes. The magnetoviscosity of these “nanotube ferrofluid” samples was studied as a function of applied magnetic field and shear frequency, and a particularly strong effect was found to be induced by viral scaffolding.

Tobacco mosaic virus (TMV) derivatives are explored currently extensively with regard to nanotechnological applications. Since certain technically desired TMV mutants may not be accessible from plants, the utility of the fission yeast Schizosaccharomyces pombe for heterologous production of TMV coat protein (CP) variants was explored, including wild-type (wt) CP and two genetically engineered mutants: TMV-CP-His6 containing a C-terminal hexahistidine (His6) tag, and TMV-CP-E50Q with enhanced lateral CP subunit interactions. After establishing expression clones and protocols for enrichment of the CP variants, their ability to reconstitute TMV-like nanostructures in the presence or absence of RNA was tested in comparison with the corresponding plant-derived CP variants, which were expressed from infectious TMV constructs. Both TMV-CP-E50Q and TMV-CP-wt yielded TMV-like rods, irrespective of the proteins' source. In contrast, His-tagged CP from plants produced only short rods in an inefficient manner, and no rods at all when expressed in yeast. This study introduces a novel approach to produce assembly competent TMV CP, but also demonstrates its limitations.

Applying a magnetic field to many ferrofluids leads to a significant increase in viscosity, but the phenomenon has yet to find technological exploitation because of the thinning caused by even weak shear flows. We have discovered that the addition of plant-virus-derived nanotubes to a commercial ferrofluid can give rise to a dramatic enhancement in magnetoviscosity and a suppression of shear thinning. The dependence of this effect on nanotube aspect ratio and surface charge, both of which were varied biotechnologically, is consistent with a "scaffolding" of magnetic particles into quasi-linear arrays. Direct support for this explanation is derived from transmission electron micrographs, which reveal a marked tendency for the magnetic nanoparticles to decorate the outside surface of the virus nanotubes.

Reference EPFL-CONF-160195View record in Web of Science Record created on 2010-11-30, modified on 2017-05-12