Equilibrium phase diagram and thermal responses of charged DNA-virus rod-suspensions at low ionic strengths.

The collective behavior of DNA is important for exploring new types of bacteria in the means of detection, which is greatly interested in the understanding of interactions between DNAs in living systems. How they self-organize themselves is a physical common phenomenon for broad ranges of thermodynamic systems. In this work, the equilibrium phase diagrams of charged chiral rods (fd viruses) at low ionic strengths (below a few mM) are provided to demonstrate both replicas of (or self-organized) twist orders and replica symmetry breaking near high concentration glass-states. By varying the ionic strengths, it appears that a critical ionic strength is obtained below 1-2 mM salt, where the twist and freezing of nematic domains diverge. Also, the microscopic relaxation is revealed by the ionic strength-dependent effective Debye screening length. At a fixed low ionic strength, the local orientations of twist are shown by two different length scales of optical pitch, in the chiral-nematic N* phase and the helical domains [Formula: see text], for low and high concentration, respectively. RSB occurs in several cases of crossing phase boundary lines in the equilibrium phase diagram of DNA-rod concentration and ionic strength, including long-time kinetic arrests in the presence of twist orders. The different pathways of PATH I, II and III are due to many-body effects of randomized orientations for charged fd rods undergoing long-range electrostatic interactions in bulk elastic medium. In addition, the thermal stability are shown for chiral pitches of the N* phase and the abnormal cooling process of a specific heat in a structural glass. Here, the concentration-driven twist-effects of charged DNA rods are explored using various experimental methods involving image-time correlation, microscopic dynamics in small angle dynamic light scattering, optical activity in second harmonic generation, and differential scanning calorimetry for the glass state.

Virus lasers for biological detection.

The selective amplification of DNA in the polymerase chain reaction is used to exponentially increase the signal in molecular diagnostics for nucleic acids, but there are no analogous techniques for signal enhancement in clinical tests for proteins or cells. Instead, the signal from affinity-based measurements of these biomolecules depends linearly on the probe concentration. Substituting antibody-based probes tagged for fluorescent quantification with lasing detection probes would create a new platform for biomarker quantification based on optical rather than enzymatic amplification. Here, we construct a virus laser which bridges synthetic biology and laser physics, and demonstrate virus-lasing probes for biosensing. Our virus-lasing probes display an unprecedented > 10,000 times increase in signal from only a 50% increase in probe concentration, using fluorimeter-compatible optics, and can detect biomolecules at sub-100 fmol mL-1 concentrations.

High Thermal Diffusivity in Thermally Treated Filamentous Virus-Based Assemblies with a Smectic Liquid Crystalline Orientation.

Polymers are generally considered thermal insulators because the amorphous arrangement of the polymeric chains reduces the mean free path of heat-conducting phonons. Recent studies reveal that individual chains of polymers with oriented structures could have high thermal conductivity, because such stretched polymeric chains effectively conduct phonons through polymeric covalent bonds. Previously, we have found that the liquid crystalline assembly composed of one of the filamentous viruses, M13 bacteriophages (M13 phages), shows high thermal diffusivity even though the assembly is based on non-covalent bonds. Despite such potential applicability of biopolymeric assemblies as thermal conductive materials, stability against heating has rarely been investigated. Herein, we demonstrate the maintenance of high thermal diffusivity in smectic liquid crystalline-oriented M13 phage-based assemblies after high temperature (150 °C) treatment. The liquid crystalline orientation of the M13 phage assemblies plays an important role in the stability against heating processes. Our results provide insight into the future use of biomolecular assemblies for reliable thermal conductive materials.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

Genetically tunable M13 phage films utilizing evaporating droplets.

This effort utilizes a genetically tunable system of bacteriophage to evaluate the effect of charge, temperature and particle concentration on biomaterial synthesis utilizing the coffee ring (CR) effect. There was a 1.6-3 fold suppression of the CR at higher temperatures while maintaining self-assembled structures of thin films. This suppression was observed in phage with charged and uncharged surface chemistry, which formed ordered and disordered assemblies respectively, indicating CR suppression is not dependent on short-range ordering or surface chemistry. Analysis of the drying process suggests weakened capillary flow at elevated temperatures caused CR suppression and could be further enhanced for controlled assembly for advanced biomaterials.

Filamentous Bacteriophage Viruses: Preparation, Magic-Angle Spinning Solid-State NMR Experiments, and Structure Determination.

Filamentous bacteriophages are elongated semi-flexible viruses that infect bacteria. They consist of a circular single-stranded DNA (ssDNA) wrapped by a capsid consisting of thousands of copies of a major coat protein subunit. Given the increasing number of discovered phages and the existence of only a handful of structures, the development of methods for phage structure determination is valuable for biophysics and structural virology. In recent years, we developed and applied techniques to elucidate the 3D atomic-resolution structures of intact bacteriophages using experimental magic-angle spinning (MAS) solid-state NMR data. The flexibility in sample preparation - precipitated homogeneous solids - and the fact that ssNMR presents no limitation on the size, weight or morphology of the system under study makes it an ideal approach to study phage systems in detail.In this contribution, we describe approaches to prepare isotopically carbon-13 and nitrogen-15 enriched intact phage samples in high yield and purity, and we present experimental MAS NMR methods to study the capsid secondary and tertiary structure, and the DNA-capsid interface. Protocols for the capsid structure determination using the Rosetta modeling software are provided. Specific examples are given from studies of the M13 and fd filamentous bacteriophage viruses.

Designing disordered materials using DNA-coated colloids of bacteriophage fd and gold.

DNA has emerged as an exciting binding agent for programmable colloidal self-assembly. Its popularity derives from its unique properties: it provides highly specific short-ranged interactions and at the same time it acts as a steric stabilizer against non-specific van der Waals and Coulomb interactions. Because complementary DNA strands are linked only via hydrogen bonds, DNA-mediated binding is thermally reversible: it provides an effective attraction that can be switched off by raising the temperature only by a few degrees. In this article we introduce a new binary system made of DNA-functionalized filamentous fd viruses of ∼880 nm length with an aspect ratio of ∼100, and 50 nm gold nanoparticles (gold NPs) coated with the complementary DNA strands. When quenching mixtures below the melt temperature Tm, at which the attraction is switched on, we observe aggregation. Conversely, above Tm the system melts into a homogenous particulate 'gas'. We present the aggregation behavior of three different gold NP to virus ratios and compare them to a gel made solely of gold NPs. In particular, we have investigated the aggregate structures as a function of cooling rate and determine how they evolve as function of time for given quench depths, employing fluorescence microscopy. Structural information was extracted in the form of an effective structure factor and chord length distributions. Rapid cooling rates lead to open aggregates, while slower controlled cooling rates closer to equilibrium DNA hybridization lead to more fine-stranded gels. Despite the different structures we find that for both cooling rates the quench into the two-phase region leads to initial spinodal decomposition, which becomes arrested. Surprisingly, although the fine-stranded gel is disordered, the overall structure and the corresponding length scale distributions in the system are remarkably reproducible. Such highly porous systems can be developed into new functional materials.

Controlling the Morphology of Organic Crystals with Filamentous Bacteriophages.

The preparation of thiamethoxam (TMX) organic crystals with high morphological uniformity was achieved by controlled aggregation-driven crystallization of primitive TMX crystals and phage using the filamentous M13 bacteriophage. The development of a regular, micrometer-sized, tetragonal-bipyramidal crystal structure was dependent on the amount of phage present. The phage appears to affect the supersaturation driving force for crystallization. The phage adsorption isotherm to TMX was well-fitted by the Satake-Yang model, which suggests a cooperative binding between neighboring phages as well as a binding of phage with the TMX crystal surface. This study shows the potential of phage additives to control the morphology and morphological uniformity of organic crystals.

Trivalent Cation Induced Bundle Formation of Filamentous fd Phages.

Bacteriophages are filamentous polyelectrolyte viral rods infecting only bacteria. In this study, we investigate the bundle formation of fd phages with trivalent cations having different ionic radii (Al(3+) , La(3+) and Y(3+) ) at various phage and counterion concentrations, and at varying bundling times. Aggregated phage bundles were detected at relatively low trivalent counterion concentrations (1 mM). Although 10 mM and 100 mM Y(3+) and La(3+) treatments formed larger and more intertwined phage bundles, Al(3+) and Fe(3+) treatments lead to the formation of networking filaments. Energy dispersive X-ray spectroscopy (EDX) analyses confirmed the presence of C, N and O peaks on densely packed phage bundles. Immunofluorescence labelling and ELISA analyses with anti-p8 antibodies showed the presence of phage filaments after bundling.

Untangling the effects of peptide sequences and nanotopographies in a biomimetic niche for directed differentiation of iPSCs by assemblies of genetically engineered viral nanofibers.

Here we report the design of a unique matrix, assembled from engineered M13 phage bionanofibers with specific cues of nanotopographies and versatile signal peptides to simulate native niche for directing the fate of induced pluripotent stem cells (iPSCs). By independently varying the peptide sequences and nanotopographies, we find that the resident iPSCs on the phage matrix are first differentiated into mesenchymal progenitor cells (MPCs), which are further differentiated into osteoblasts in the absence of osteogenic supplements due to the elongation induced by phage nanofibers. The phage-based matrix represents not only a biomimetic stem cell niche enabling independently varying biochemical and biophysical cues in one system but also a substrate for generating a safe and efficient cell source for tissue engineering.

M13 virus-directed synthesis of nanostructured metal oxides for lithium-oxygen batteries.

Transition metal oxides are promising electrocatalysts for both water oxidations and metal-air batteries. Here, we report the virus-mediated synthesis of cobalt manganese oxide nanowires (NWs) to fabricate high capacity Li-O2 battery electrodes. Furthermore, we hybridized Ni nanoparticles (NPs) on bio Co3O4 NWs to improve the round trip efficiency as well as the cycle life of Li-O2 batteries. This biomolecular directed synthesis method is expected to provide a selection platform for future energy storage electrocatalysts.

Responsive 3D microstructures from virus building blocks.

Fabrication of 3D biological structures reveals dynamic response to external stimuli. A liquid-crystalline bridge extrusion technique is used to generate 3D structures allowing the capture of Rayleigh-like instabilities, facilitating customization of smooth, helical, or undulating periodic surface textures. By integrating intrinsic biochemical functionality and synthetic components into controlled structures, this strategy offers a new form of adaptable materials.

Assembly of viral hydrogels for three-dimensional conducting nanocomposites.

M13 bacteriophages act as versatile scaffolds capable of organizing single-walled carbon nanotubes and fabricating three-dimensional conducting nanocomposites. The morphological, electrical, and electrochemical properties of the nanocomposites are presented, as well as its ability to disperse and utilize single-walled carbon nanotubes effectively.

Virus templated gold nanocube chain for SERS nanoprobe.

A M13 virus based SERS nanoprobe is presented. Gold nanocubes closely aligned into chains along the length of the virus intensify Raman signals of various reporter molecules serving as specific labels. An antibody is expressed at one end to detect the analyte. This new SERS nanoprobe holds promise for infinitesimal and multiplexed detection of any antigen.

Highly sensitive hydrogen sulfide (H2S) gas sensors from viral-templated nanocrystalline gold nanowires.

A facile, site-specific viral-templated assembly method was used to fabricate sensitive hydrogen sulfide (H2S) gas sensors at room temperature. A gold-binding M13 bacteriophage served to organize gold nanoparticles into linear arrays which were used as seeds for subsequent nanowire formation through electroless deposition. Nanowire widths and densities within the sensors were modified by electroless deposition time and phage concentration, respectively, to tune device resistance. Chemiresistive H2S gas sensors with superior room temperature sensing performance were produced with sensitivity of 654%/ppm(v), theoretical lowest detection limit of 2 ppb(v), and 70% recovery within 9 min for 0.025 ppm(v). The role of the viral template and associated gold-binding peptide was elucidated by removing organics using a short O2 plasma treatment followed by an ethanol dip. The template and gold-binding peptide were crucial to electrical and sensor performance. Without surface organics, the resistance fell by several orders of magnitude, the sensitivity dropped by more than a factor of 100 to 6%/ppm(v), the lower limit of detection increased, and no recovery was detected with dry air flow. Viral templates provide a novel, alternative fabrication route for highly sensitive, nanostructured H2S gas sensors.

Assembly of a bacteriophage-based template for the organization of materials into nanoporous networks.

M13 bacteriophages are assembled via a covalent layer-by-layer process to form a highly nanoporous network capable of organizing nanoparticles and acting as a scaffold for templating metal-oxides. The morphological and optical properties of the film itself are presented as well as its ability to organize and disperse metal nanoparticles.

Recombinant bacteriophages as gold binding bio-templates.

Bacteriophages are nano-sized virion particles infecting bacteria. In this study, it is shown that metal binding properties of filamentous fd-bacteriophages can be enhanced by genetic engineering. Quartz crystal microbalance (QCM) analyses, UV-vis absorption spectra measurements and scanning electron microscopy (SEM) imaging revealed that expression of MMM short amino acid sequence on major coat protein p8 facilitates recombinant MMM-phage binding to Au surfaces and nanoparticles (NPs) via gold-sulfur (AuS) interaction. Electroless deposition of Au particles on phage assemblies was investigated upon chemical reduction reaction with NaBH4 at different HAuCl4 precursor concentrations. Energy dispersive X-ray spectroscopy (EDX) measurements confirmed the presence of Au on both AuNP decorated and chemically metallized phage structures. Further studies on patterning and controlled immobilization of recombinant bacteriophages on specific surfaces may contribute to bio-templated nanowire development field and biosensor application studies.

Phage-directed synthesis of copper sulfide: structural and optical characterization.

The growth of crystalline copper sulfide using a viral template was investigated using sequential incubation in CuCl2 and Na2S precursors. Non-specific electrostatic attraction between a genetically-modified M13 bacteriophage and copper cations in the CuCl2 precursor caused phage agglomeration and bundle formation. Following the addition of Na2S, polydisperse nanocrystals 2-7 nm in size were found along the length of the viral scaffold. The structure of the copper sulfide material was identified as cubic anti-fluorite type Cu1.8S, space group Fm3[overline]m. Strong interband absorption was observed within the ultraviolet to visible range with an onset near 800 nm. Furthermore, free carrier absorption, associated with the localized surface plasmon resonance of the copper sulfide nanocrystals, was seen in the near infrared with absorbance maxima at 1060 nm and 3000 nm, respectively.

Evolutionary screening and adsorption behavior of engineered M13 bacteriophage and derived dodecapeptide for selective decoration of gold interfaces.

There is a growing interest in identifying biomacromolecules such as proteins and peptides to functionalize metallic surfaces through noncovalent binding. One method for functionalizing materials without fundamentally changing their inherent structure is using biorecognition moieties. Here, we proved a general route to select a biomolecule adhesive motif for surface functionalization by comprehensively screening phage displayed peptides. In particular, we selected a genetically engineered M13 bacteriophage and a linear dodecapeptide derived from its pIII domain for recognizing gold surfaces in a specific and selective manner. In the phage context, we demonstrated the adhesive motif was capable to adsorb on gold in a preferential way with a morphological and viscoelastic signature of the adsorbed layer as evidenced by QCM-D and AFM investigations. Out of the phage context, the linear dodecapeptide is reproducibly found to adhere to the gold surface, and by quantitative SPR measurements, high affinity constants (K(eq)~10(6)M(-1), binding energy ~-8 kcal/mol) were determined. We proved that the interactions occurring at gold interface were mainly hydrophobic as a consequence of high frequency of hydrophobic residues in the peptide sequence. Moreover, by CD, molecular dynamics and steered molecular dynamics, we demonstrated that the molecular flexibility only played a minor role in the peptide adsorption. Such noncovalent but specific modification of inorganic surfaces through high affinity biomolecule adsorption represents a general strategy to modulate the functionality of multipurpose metallic surfaces.