Plant virus-based materials for biomedical applications: Trends and prospects.

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.

Precise Assembly of Genetically Functionalized Magnetosomes and Tobacco Mosaic Virus Particles Generates a Magnetic Biocomposite.

Magnetosomes represent magnetic nanoparticles with unprecedented characteristics. Both their crystal morphology and the composition of the enveloping membrane can be manipulated by genetic means, allowing the display of functional moieties on the particle surface. In this study, we explore the generation of a new biomaterial assembly by coupling magnetosomes with tobacco mosaic virus (TMV) particles, both functionalized with complementary recognition sites. TMV consists of single-stranded RNA encapsidated by more than 2100 coat proteins, which enable chemical modification via functional groups. Incubation of EmGFP- or biotin-decorated TMV particles with magnetosomes genetically functionalized with GFP-binding nanobodies or streptavidin, respectively, results in the formation of magnetic, mesoscopic, strand-like biocomposites. TMV facilitates the agglomeration of magnetosomes by providing a scaffold. The size of the TMV-magnetosome mesostrands can be adjusted by varying the TMV-magnetosome particle ratios. The versatility of this novel material combination is furthermore demonstrated by coupling magnetosomes and terminal, 5'-functionalized TMV particles with high molecular precision, which results in "drumstick"-like TMV-magnetosome complexes. In summary, our approaches provide promising strategies for the generation of new biomaterial assemblies that could be used as scaffold for the introduction of further functionalities, and we foresee a broad application potential in the biomedical and biotechnological field.

Bioinspired Silica Mineralization on Viral Templates.

Plant virus capsids are attractive entities for nanotechnological applications because of their variation in shape and natural assembly ability. This chapter describes the production and modification of three differently shaped plant virus capsids for silica mineralization purposes. The chosen plant viruses exhibit either an icosahedral (cowpea mosaic virus, CPMV), or a flexuous rod-like structure (potato virus X, PVX), or a rigid rod-like shape (tobacco mosaic virus, TMV), and are well-known and frequently used plant viruses for biotechnological applications. We describe the production (including genetic or chemical modification) and purification of the plant viruses or of empty virus-like particles in the case of CPMV, as well as the characterization of these harvested templates. The mineralization procedures and differences in the protocols specific to the distinct viruses are described, and the analyses of the mineralization results are explained.

Electrochemically-Driven Insertion of Biological Nanodiscs into Solid State Membrane Pores as a Basis for "Pore-In-Pore" Membranes.

Nanoporous membranes are of increasing interest for many applications, such as molecular filters, biosensors, nanofluidic logic and energy conversion devices. To meet high-quality standards, e.g., in molecular separation processes, membranes with well-defined pores in terms of pore diameter and chemical properties are required. However, the preparation of membranes with narrow pore diameter distributions is still challenging. In the work presented here, we demonstrate a strategy, a "pore-in-pore" approach, where the conical pores of a solid state membrane produced by a multi-step top-down lithography procedure are used as a template to insert precisely-formed biomolecular nanodiscs with exactly defined inner and outer diameters. These nanodiscs, which are the building blocks of tobacco mosaic virus-deduced particles, consist of coat proteins, which self-assemble under defined experimental conditions with a stabilizing short RNA. We demonstrate that the insertion of the nanodiscs can be driven either by diffusion due to a concentration gradient or by applying an electric field along the cross-section of the solid state membrane. It is found that the electrophoresis-driven insertion is significantly more effective than the insertion via the concentration gradient.

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO2 mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

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. 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.

Peptide-equipped tobacco mosaic virus templates for selective and controllable biomineral deposition.

The coating of regular-shaped, readily available nanorod biotemplates with inorganic compounds has attracted increasing interest during recent years. The goal is an effective, bioinspired fabrication of fiber-reinforced composites and robust, miniaturized technical devices. Major challenges in the synthesis of applicable mineralized nanorods lie in selectivity and adjustability of the inorganic material deposited on the biological, rod-shaped backbones, with respect to thickness and surface profile of the resulting coating, as well as the avoidance of aggregation into extended superstructures. Nanotubular tobacco mosaic virus (TMV) templates have proved particularly suitable towards this goal: Their multivalent protein coating can be modified by high-surface-density conjugation of peptides, inducing and governing silica deposition from precursor solutions in vitro. In this study, TMV has been equipped with mineralization-directing peptides designed to yield silica coatings in a reliable and predictable manner via precipitation from tetraethoxysilane (TEOS) precursors. Three peptide groups were compared regarding their influence on silica polymerization: (i) two peptide variants with alternating basic and acidic residues, i.e. lysine-aspartic acid (KD) x motifs expected to act as charge-relay systems promoting TEOS hydrolysis and silica polymerization; (ii) a tetrahistidine-exposing polypeptide (CA4H4) known to induce silicification due to the positive charge of its clustered imidazole side chains; and (iii) two peptides with high ZnO binding affinity. Differential effects on the mineralization of the TMV surface were demonstrated, where a (KD) x charge-relay peptide (designed in this study) led to the most reproducible and selective silica deposition. A homogenous coating of the biotemplate and tight control of shell thickness were achieved.