Prototype Protein Assembly as Scaffold for Time-Resolved Fluoroimmuno Assays

Abstract

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.

Crosslinking of viral nanoparticles with “clickable” fluorescent crosslinkers at the interface

“…ESI HRMS spectra for compounds were recorded using Micromass Q-TOF. TYMV was purified from infected Chinese cabbage leaves following the procedure as described [43]. Chemical modification of TYMV with the desired compounds and its characterization was done using the established protocols as described [42].…”

“…N-(propargyl)-2-methylbenzothiazolium bromide 1 was synthesized according to a known protocol [41]. Protein gel and Western blotting were performed on a 15% SDS-PAGE gel following the standard protocols [43]. The primary TYMV polyclonal antibody was provided by Prof. Theo Dreher (Oregon State University).…”

“…A plant virus, turnip yellow mosaic virus (TYMV), was used as the model to demonstrate the reaction potential of these dyes in crosslinking two particles. Recent studies indicate that TYMV can be employed as a great tool in drug delivery, imaging and tissue engineering applications [6,[42][43][44][45][46]. This symmetric bionanoparticle (BNP) could self-assemble at the liquid-liquid interface to yield stable emulsion systems and highly ordered long range arrays at the flat interface [44].…”

Viruses and virus-like protein assemblies—Chemically programmable nanoscale building blocks

“…2). The systematic characterization of cowpea mosaic virus (CPMV) [40 44], along with studies of cowpea chlorotic mottle virus (CCMV) [45], bacteriophage MS2 [46], heat shock protein [18], tobacco mosaic virus (TMV) [47], and turnip yellow mosaic virus (TYMV) [48], have shed light on the unique chemical reactivities and physical properties of these individual BNPs. These initial discoveries have been integred to design BNPs as nano-scaffolds for drug delivery, bioimaging, biomineralization, and even tissue engineering.…”

“…These reagents have been previously used in the derivatization of carboxylic groups of ferritins with long chain aliphatic amines, resulting in stable, hydrophobic macromolecules [53,54]. In recent years, systematic characterizations implemented with EDC coupling reactions have been conducted on various plant viruses (CPMV, TYMV, CCMV, and TMV) [45,47,48,55]. Schlick and co-authors reported efficient coupling via EDC in the presence of 1-hydroxybenzotriazole (HOBT) [47].…”