Chemoselective modification of turnip yellow mosaic virus by Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction and its application in cell binding.

Turnip yellow mosaic virus (TYMV) is an icosahedral plant virus with a diameter of 28-30 nm that can be isolated in gram quantities from turnip or Chinese cabbage inexpensively. In this study, TYMV combined with spatially addressable surface chemistries was selected as a prototype bionanoparticle for modulating patterns of cell adhesion, morphology, and proliferation. We exploited the chemical reactivity of TYMV using the mild conditions of Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction, the best example of "click" chemistry. Oligo-ethylene glycol (OEG) short chain, coumarintriazole, and RGD-containing peptide were grafted on the surface of TYMV via carbodiimide activation and CuAAC reaction. The bioconjugation to intact viral particles was confirmed by MS, TEM, FPLC, and SDS-PAGE with fluorescence visualization analysis. Therefore, this method is a generally useful means of incorporating various types of functionalities onto the TYMV surface. Further studies were done to learn the behavior of NIH-3T3 fibroblast cells on the modified or unmodified TYMV surfaces. OEG-modified TYMV surfaces retarded cell attachment and growth, while cell adhesion, spreading, and proliferation were dramatically enhanced on RGD-modified TYMV surfaces. Compared with RGD immobilized 3-aminopropyltriethoxysilane-coated glass surface, the cells are more ready to spread fully and proliferate on TYMV-RGD coated surface, which thus provides a more cell-friendly environment with nanometer-scale surface features. This illustrates the potential application of plant virus based materials in tissue engineering, drug delivery, and biosensing.

Cowpea mosaic virus capsid: a promising carrier for the development of carbohydrate based antitumor vaccines.

Immunotherapy targeting tumor cell surface carbohydrates is a promising approach for cancer treatment. However, the low immunogenecity of carbohydrates presents a formidable challenge. We describe here the enhancement of carbohydrate immunogenicity by an ordered display on the surface of the cowpea mosaic virus (CPMV) capsid. The Tn glycan, which is overexpressed on numerous cancer cell surfaces, was selected as the model antigen for our study. Previously it has been shown that it is difficult to induce a strong T cell-dependent immune response against the monomeric form of Tn presented in several ways on different carriers. In this study, we first synthesized Tn antigens derivatized with either a maleimide or a bromoacetamide moiety that was conjugated selectively to a cysteine mutant of CPMV. The glycoconjugate was then injected into mice and pre- and post-immune antibody levels in the mice sera were measured by enzyme-linked immunosorbant assays. High total antibody titers and, more importantly, high IgG titers specific for Tn were obtained in the post-immune day 35 serum, suggesting the induction of T cell-dependent antibody isotype switching by the glycoconjugate. The antibodies generated were able to recognize Tn antigens presented in their native conformations on the surfaces of both MCF-7 breast cancer cells and the multidrug resistant breast cancer cell line NCI-ADR RES. These results suggest that the CPMV capsid can greatly enhance the immunogenicity of weak antigens such as Tn and this can provide a promising tool for the development of carbohydrate based anti-cancer vaccines.

Prototype protein assembly as scaffold for time-resolved fluoroimmuno assays.

Turnip yellow mosaic virus (TYMV) is an icosahedral plant virus with an average diameter of 28 nm and can be isolated in gram quantities from turnip or Chinese cabbage inexpensively. In this study, it was selected as a prototype bionanoparticle for time-resolved fluoroimmuno assay (TRFIA). Two types of reactive amino acid residues were employed to anchor luminescent terbium complexes and biotin groups based on orthogonal chemical reactions. While terbium complexes were used as luminescent signaling groups, biotin motifs acted as a model ligand for protein binding. The bioconjugation results were confirmed by MS and Western blot analysis. Steady-state and time-resolved luminescence study of the dual-modified viruses demonstrated that the spectroscopic properties of the Tb complex are unperturbed by the labeling procedure. The dual-modified particle was probed by fluorescence resonance energy transfer (FRET) experiments using avidin labeled with an Alexa488 fluorophore, which bound to the biotin on the surface of the particle, as an energy acceptor, and terbium complexes as an energy donor. The emission and excitation spectra of the dual-labeled TYMV particle displayed residual virus fluorescence and Tb luminescence upon ligand-centered excitation. The Tb luminescence lifetime was 1.62 ms and could be effectively fitted with a single-exponential behavior. In the TRFIA, an efficient transfer of 66% was observed, and the calculation using the Förster radius of 41 A allowed for an estimation of the average donor-acceptor distance of 36 A. Our studies show that the two reactive sites can communicate with each other on the surface of a nanoscale biological assembly. In particular, the ligand-receptor binding (biotin and avidin in this paper) was not interfered with when anchored to the surface of TYMV. Therefore, as a prototype of polyvalent bionanoparticles, TYMV can be used as scaffold for sensor development with TRFIA.

Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.

Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the two-dimensional self-assembly of viruses and VLPs, the icosahedral turnip yellow mosaic virus (TYMV) was investigated as a potential building block in our study. TYMV is an icosahedral plant virus with an average diameter of 28 nm that can be isolated inexpensively in gram quantities from turnips or Chinese cabbage. There are 180 coat protein subunits per TYMV capsid. The conventional N-hydroxysuccinimide-mediated amidation reaction was employed for the chemical modification of the viral capsid. Tryptic digestion with sequential MALDI-TOF MS analysis identified that the amino groups of K32 of the flexible N-terminus made the major contribution for the reactivity of TYMV toward N-hydroxysuccinimide ester (NHS) reagents. The reactivity was also monitored with UV-vis absorbance and fluorescence, which revealed that approximately 60 lysines per particle could be addressed. We hypothesized that the flexible A chain contains the reactive lysine because the crystal structure of TYMV has shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the Lys-32 located. In addition, about 90 to 120 carboxyl groups, located in the most exposed sequence, could be modified with amines catalyzed with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulfo-NHS. TYMV was stable to a wide range of reaction conditions and maintained its integrity after the chemical conjugations. Therefore, it can potentially be employed as a reactive scaffold for the display of a variety of materials for applications in many areas of nanoscience.

A fluorogenic 1,3-dipolar cycloaddition reaction of 3-azidocoumarins and acetylenes.

Copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of nonfluorescent 3-azidocoumarins and terminal alkynes afforded intense fluorescent 1,2,3-triazole products. The mild condition of this reaction allowed us to construct a large library of pure fluorescent coumarin dyes. Since both azide and alkyne are quite inert to biological systems, this reaction has potential in bioconjugation and bioimaging applications. [reaction: see text]