A highly sensitive fluorescence sensor for tobacco mosaic virus RNA based on DSN cycle and ARGET ATRP double signal amplification.

Early and sensitive detection of tobacco mosaic virus (TMV) is of great significance for improving crop yield and protecting germplasm resources. Herein, we constructed a novel fluorescence sensor to detect TMV RNA (tRNA) through double strand specific nuclease (DSN) cycle and activator regenerative electron transfer atom transfer radical polymerization (ARGET ATRP) dual signal amplification strategy. The hairpin DNA complementarily paired with tRNA was used as a recognition unit to specifically capture tRNA. By the double-stranded DNA hydrolyzed with DSN, tRNA is released to open more hairpin DNA, and more complementary DNA (cDNA) is bound to the surface of the magnetic beads (MBs) to achieve the first amplification. After binding with the initiator, the cDNA employed ARGET ATRP to attach more fluorescent signal molecules to the surface of MBs, thus achieving the second signal amplification. Under the optimal experimental conditions, the logarithm of fluorescence intensity versus tRNA concentration showed a good linear relationship in the range of 0.01-100 pM, with a detection limit of 1.03 fM. The limit of detection (LOD) was calculated according to LOD = 3 N/S. Besides, the sensor showed good reproducibility and stability, which present provided new method for early and highly sensitive detection for plant viruses.

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

In Situ Insights into the Nucleation and Growth Mechanisms of Gold Nanoparticles on Tobacco Mosaic Virus.

Biotemplated syntheses have emerged as an efficient strategy to control the assembly of metal nanoparticles (NPs) and generate promising plasmonic properties for sensing or biomedical applications. However, understanding the nucleation and growth mechanisms of metallic nanostructures on biotemplate is an essential prerequisite to developing well-controlled nanotechnologies. Here, we used liquid cell Transmission Electron Microscopy (TEM) to reveal how the formation kinetics of gold NPs affects their size and density on Tobacco Mosaic Virus (TMV). These in situ insights are used as a guideline to optimize bench-scale synthesis with the possibility to homogenize the coverage and tune the density of gold NPs on TMV. In line with in situ TEM observations, fluorescence spectroscopy confirms that the nucleation of NPs occurs on the virus capsid rather than in solution. The proximity of gold NPs on TMV allows shifting the plasmonic resonance of the assembly in the biological window.

Delivery of Nematicides Using TMGMV-Derived Spherical Nanoparticles.

Spherical nanoparticles (SNPs) from tobacco mild green mosaic virus (TMGMV) were developed and characterized, and their application for agrochemical delivery was demonstrated. Specifically, we set out to develop a platform for pesticide delivery targeting nematodes in the rhizosphere. SNPs were obtained by thermal shape-switching of the TMGMV. We demonstrated that cargo can be loaded into the SNPs during thermal shape-switching, enabling the one-pot synthesis of functionalized nanocarriers. Cyanine 5 and ivermectin were encapsulated into SNPs to achieve 10% mass loading. SNPs demonstrated good mobility and soil retention slightly higher than that of TMGMV rods. Ivermectin delivery to Caenorhabditis elegans using SNPs was determined after passing the formulations through soil. Using a gel burrowing assay, we demonstrate the potent efficacy of SNP-delivered ivermectin against nematodes. Like many pesticides, free ivermectin is adsorbed in the soil and did not show efficacy. The SNP nanotechnology offers good soil mobility and a platform technology for pesticide delivery to the rhizosphere.

Capacitive field-effect biosensor modified with a stacked bilayer of weak polyelectrolyte and plant virus particles as enzyme nanocarriers.

This work presents a new approach for the development of field-effect biosensors based on an electrolyte-insulator-semiconductor capacitor (EISCAP) modified with a stacked bilayer of weak polyelectrolyte and tobacco mosaic virus (TMV) particles as enzyme nanocarriers. With the aim to increase the surface density of virus particles and thus, to achieve a dense immobilization of enzymes, the negatively charged TMV particles were loaded onto the EISCAP surface modified with a positively charged poly(allylamine hydrochloride) (PAH) layer. The PAH/TMV bilayer was prepared on the Ta2O5-gate surface by means of layer-by-layer technique. The bare and differently modified EISCAP surfaces were physically characterized by fluorescence microscopy, zeta-potential measurements, atomic force microscopy and scanning electron microscopy. Transmission electron microscopy was used to scrutinize the PAH effect on TMV adsorption in a second system. Finally, a highly sensitive TMV-assisted EISCAP antibiotics biosensor was realized by immobilizing the enzyme penicillinase onto the TMV surface. This PAH/TMV bilayer-modified EISCAP biosensor was electrochemically characterized in solutions with different penicillin concentrations via capacitance-voltage and constant-capacitance methods. The biosensor possessed a mean penicillin sensitivity of 113 mV/dec in a concentration range from 0.1 mM to 5 mM.

Design, Synthesis, Biological Activity Evaluation and Action Mechanism of Myricetin Derivatives Containing Thiazolebisamide.

The plant diseases caused by a variety of pathogens such as viruses, bacteria and fungi pose a great threat to global food production and food safety. Therefore, the search for green, efficient and pollution-free pesticides has become an important task. In this article, 23 myricetin derivatives containing thiazolebisamides active groups have been designed and synthesized. Their activities were evaluated by performing in vitro antibacterial and in vivo antiviral assays, microscale thermophoresis (MST) and molecular docking assays. The results of in vivo antiviral assays showed that compounds A4 and A23 exhibited good antiviral activity with EC50 values of 79.0 and 54.1 µg/mL for therapeutic activity and 103.3 and 91.2 µg/mL for protective activity, respectively. The dissociation constants (Kd) values of compounds A4 and A23 against TMV-CP were 0.021 and 0.018 µM, respectively, determined by microscale thermophoresis (MST), which were much smaller than those of the commercial drug ningnanmycin (NNM), which were 2.84 µM. The interaction of compounds A4, A23 with TMV-CP was further verified at the molecular level. In addition, in vitro antifungal assays of this series of compounds showed that they exhibited some inhibitory activity against a variety of fungi, especially against the phytophthora capsici. Among them, A13 and A20 showed similar inhibitory activity to the control drug azoxystrobin at 100 µg/mL against the phytophthora capsici.

Characterizing Heterogeneous Mixtures of Assembled States of the Tobacco Mosaic Virus Using Charge Detection Mass Spectrometry.

The tobacco mosaic viral capsid protein (TMV) is a frequent target for derivatization for myriad applications, including drug delivery, biosensing, and light harvesting. However, solutions of the stacked disk assembly state of TMV are difficult to characterize quantitatively due to their large size and multiple assembled states. Charge detection mass spectrometry (CDMS) addresses the need to characterize heterogeneous populations of large protein complexes in solution quickly and accurately. Using CDMS, previously unobserved assembly states of TMV, including 16-monomer disks and odd-numbered disk stacks, have been characterized. We additionally employed a peptide-protein conjugation reaction in conjunction with CDMS to demonstrate that modified TMV proteins do not redistribute between disks. Finally, this technique was used to discriminate between protein complexes of near-identical mass but different configurations. We have gained a greater understanding of the behavior of TMV, a protein used across a broad variety of fields and applications, in the solution state.

Static Disorder has Dynamic Impact on Energy Transport in Biomimetic Light-Harvesting Complexes.

Despite extensive studies, many questions remain about what structural and energetic factors give rise to the remarkable energy transport efficiency of photosynthetic light-harvesting protein complexes, owing largely to the inability to synthetically control such factors in these natural systems. Herein, we demonstrate energy transfer within a biomimetic light-harvesting complex consisting of identical chromophores attached in a circular array to a protein scaffold derived from the tobacco mosaic virus coat protein. We confirm the capability of energy transport by observing ultrafast depolarization in transient absorption anisotropy measurements and a redshift in time-resolved emission spectra in these complexes. Modeling the system with kinetic Monte Carlo simulations recapitulates the observed anisotropy decays, suggesting an inter-site hopping rate as high as 1.6 ps-1. With these simulations, we identify static disorder in orientation, site energy, and degree of coupling as key remaining factors to control to achieve long-range energy transfer in these systems. We thereby establish this system as a highly promising, bottom-up model for studying long-range energy transfer in light-harvesting protein complexes.

Cisplatin-Loaded Tobacco Mosaic Virus for Ovarian Cancer Treatment.

Ovarian cancer is the foremost cause of gynecological cancer and a major cause of cancer death in women. Treatment for advanced stage is surgical debulking followed by chemotherapy; however, most patients relapse with more aggressive and therapy-resistant tumors. There is a need to develop drug delivery approaches to deliver platinum therapies to tumors to increase efficacy while maintaining safety. Toward this goal, we utilized the protein nanotubes from the plant virus, tobacco mosaic virus (TMV), as a drug carrier. Specifically, the nanochannel of TMV was loaded with the active dication form of cisplatin (cisPt2+), making use of the negatively charged Glu acid side chains that line the interior channel of TMV. We achieved a loading efficiency with ∼2700 cisPt2+ per TMV; formulation stability was established with drug complexes stably loaded into the carrier for 2 months under refrigerated storage. TMV-cisPt maintained its efficacy against ovarian tumor cells with an IC50 of ∼40 µM. TMV-cisPt exhibited superior efficacy vs free cisPt in ovarian tumor mouse models using intraperitoneal ID8-Defb29/Vegf-a-Luc (mouse) tumors and subcutaneous A2780 (human) xenografts. TMV-cisPt treatment led to reduced tumor burden and increased survival. Using ID8-Defb29/Vegf-a-Luc-bearing C57BL/6 mice, we also noted reduced tumor growth when animals were treated with TMV alone, which may indicate antitumor immunity induced by the immunomodulatory nature of the plant virus nanoparticle. Biodistribution studies supported the efficacy data, showing increased cisPt accumulation within tumors when delivered via the TMV carrier vs free cisPt administration. Finally, good safety profiles were noted. The study highlights the potential of TMV as a drug carrier against cancer and points to the opportunity to explore plant viruses as chemo-immuno combination cancer therapeutics.

Biomolecular Self-Assembly of Nanorings on a Viral Protein Template.

Although there have been many advances in synthesizing nanoparticles, their assembly into deterministic and controllable patterns remains a major challenge. Biological systems operate at the nanoscale, building structural components with great chemical specificity that enable the processes of life. By adapting them to our needs, it is possible to utilize well-defined and well-controlled scaffolds to produce materials with novel properties resulting from precise ordering on the nanoscale. This approach uses spatial arrangement instead of nanoparticle size, shape, or composition to control material properties through the collective interactions between neighboring nanoparticles. Here, we demonstrate the use of tobacco mosaic virus (TMV) coat protein as a template to self-assemble plasmonic nanoparticles. Surface plasmons are resonant oscillations in the free electrons of a metal that are excited through interaction with light. These plasmonic oscillations can couple together, giving rise to more complex modes like plasmonic ring resonances that can be used to tune the response to incident light. By exploiting the self-assembling properties and chemical addressability of TMV coat protein, we can utilize site-directed mutagenesis and bioconjugation strategies to produce highly symmetrical plasmonic nanorings, as evidenced by transmission electron microscopy (TEM). Thus, we show the utility of viral proteins in designing and assembling nanostructured building blocks for advanced materials.

Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus.

Tobacco mild green mosaic virus (TMGMV) is a plant virus closely related to Tobacco mosaic virus (TMV), sharing many of its structural and chemical features. These rod-shaped viruses, comprised of 2130 identical coat protein subunits, have been utilized as nanotechnological platforms for a myriad of applications, ranging from drug delivery to precision agriculture. This versatility for functionalization is due to their chemically active external and internal surfaces. While both viruses are similar, they do exhibit some key differences in their surface chemistry, suggesting the reactive residue distribution on TMGMV should not overlap with TMV. In this work, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify the N-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies. We also report that cargo can be loaded to the solvent-exposed N-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming.

One-Step Supramolecular Multifunctional Coating on Plant Virus Nanoparticles for Bioimaging and Therapeutic Applications.

Plant viral nanoparticles (plant VNPs) are promising biogenetic nanosystems for the delivery of therapeutic, immunotherapeutic, and diagnostic agents. The production of plant VNPs is simple and highly scalable through molecular farming in plants. Some of the important advances in VNP nanotechnology include genetic modification, disassembly/reassembly, and bioconjugation. Although effective, these methods often involve complex and time-consuming multi-step protocols. Here, we report a simple and versatile supramolecular coating strategy for designing functional plant VNPs via metal-phenolic networks (MPNs). Specifically, this method gives plant viruses [e.g., tobacco mosaic virus (TMV), cowpea mosaic virus, and potato virus X] additional functionalities including photothermal transduction, photoacoustic imaging, and fluorescent labeling via different components in MPN coating [i.e., complexes of tannic acid (TA), metal ions (e.g., Fe3+, Zr4+, or Gd3+), or fluorescent dyes (e.g., rhodamine 6G and thiazole orange)]. For example, using TMV as a viral substrate by choosing Zr4+-TA and rhodamine 6G, fluorescence is observed peaking at 555 nm; by choosing Fe3+-TA coating, the photothermal conversion efficiency was increased from 0.8 to 33.2%, and the photoacoustic performance was significantly improved with a limit of detection of 17.7 µg mL-1. We further confirmed that TMV@Fe3+-TA nanohybrids show good cytocompatibility and excellent cell-killing performance in photothermal therapy with 808 nm irradiation. These findings not only prove the practical benefits of this supramolecular coating for designing multifunctional and biocompatible plant VNPs but also bode well for using such materials in a variety of plant virus-based theranostic applications.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

Enzyme Activated Gold Nanoparticles for Versatile Site-Selective Bioconjugation.

A new enzymatic method is reported for constructing protein- and DNA-AuNP conjugates. The strategy relies on the initial functionalization of AuNPs with phenols, followed by activation with the enzyme tyrosinase. Using an oxidative coupling reaction, the activated phenols are coupled to proteins bearing proline, thiol, or aniline functional groups. Activated phenol-AuNPs are also conjugated to a small molecule biotin and commercially available thiol-DNA. Advantages of this approach for AuNP bioconjugation include: (1) initial formation of highly stable AuNPs that can be selectively activated with an enzyme, (2) the ability to conjugate either proteins or DNA through a diverse set of functional handles, (3) site-specific immobilization, and (4) facile conjugation that is complete within 2 h at room temperature under aqueous conditions. The enzymatic oxidative coupling on AuNPs is applied to the construction of tobacco mosaic virus (TMV)-AuNP conjugates, and energy transfer between the AuNPs and fluorophores on TMV is demonstrated.

Two particle-picking procedures for filamentous proteins: SPHIRE-crYOLO filament mode and SPHIRE-STRIPER.

Structure determination of filamentous molecular complexes involves the selection of filaments from cryo-EM micrographs. The automatic selection of helical specimens is particularly difficult, and thus many challenging samples with issues such as contamination or aggregation are still manually picked. Here, two approaches for selecting filamentous complexes are presented: one uses a trained deep neural network to identify the filaments and is integrated in SPHIRE-crYOLO, while the other, called SPHIRE-STRIPER, is based on a classical line-detection approach. The advantage of the crYOLO-based procedure is that it performs accurately on very challenging data sets and selects filaments with high accuracy. Although STRIPER is less precise, the user benefits from less intervention, since in contrast to crYOLO, STRIPER does not require training. The performance of both procedures on Tobacco mosaic virus and filamentous F-actin data sets is described to demonstrate the robustness of each method.

Programming Dynamic Assembly of Viral Proteins with DNA Origami.

Biomolecular assembly in biological systems is typically a complex dynamic process regulated by the exchange of molecular information between biomolecules such as proteins and nucleic acids. Here, we demonstrate a nucleic-acid-based system that can program the dynamic assembly process of viral proteins. Tobacco mosaic virus (TMV) genome-mimicking RNA is anchored on DNA origami nanostructures via hybridization with a series of DNA strands which also function as locks that prevent the packaging of RNA by the TMV proteins. The selective, sequential releasing of the RNA via toehold-mediated strand displacement allows us to program the availability of RNA and subsequently the TMV growth in situ. Furthermore, the programmable dynamic assembly of TMV on DNA templates also enables the production of new DNA-protein hybrid nanostructures, which are not attainable by using previous assembly methods.

Secondary modification of oxidatively-modified proline N-termini for the construction of complex bioconjugates.

A convenient two-step method is reported for the ligation of alkoxyamine- or hydrazine-bearing cargo to proline N-termini. Using this approach, bifunctional proline N-terminal bioconjugates are constructed and proline N-terminal proteins are immobilized.

Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization.

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense "grains" on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzyme-linked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

FiXR: a framework to reconstruct fiber cross-sections from X-ray fiber diffraction experiments.

Ab initio reconstruction methods have revolutionized the capabilities of small-angle X-ray scattering (SAXS), allowing the data-driven discovery of previously unknown molecular conformations, exploiting optimization heuristics and assumptions behind the composition of globular molecules. While these methods have been successful for the analysis of small particles, their impact on fibrillar assemblies has been more limited. The micrometre-range size of these assemblies and the complex interaction of their periodicities in their scattering profiles indicate that the discovery of fibril structures from SAXS measurements requires novel approaches beyond extending existing tools for molecular discovery. In this work, it is proposed to use SAXS measurements, together with diffraction theory, to infer the electron distribution of the average cross-section of a fiber. This cross-section is modeled as a discrete electron density with continuous support, allowing representations beyond binary distributions. Additional constraints, such as non-negativity or smoothness/connectedness, can also be added to the framework. The proposed approach is tested using simulated SAXS data from amyloid ß fibril models and using measured data of Tobacco mosaic virus from SAXS experiments, recovering the geometry and density of the cross-sections in all cases. The approach is further tested by analyzing SAXS data from different amyloid ß fibril assemblies, with results that are in agreement with previously proposed models from cryo-EM measurements. The limitations of the proposed method, together with an analysis of the robustness of the method and the combination with different experimental sources, are also discussed.