Abstract
Hepatitis E Virus-like particles self-assemble in to noninfectious nanocapsids that are resistant to proteolytic/acidic mucosal delivery conditions. Previously, the nanocapsid was engineered to specifically bind and enter breast cancer cells, where successful tumor targeting was demonstrated in animal models. In the present study, the nanocapsid surface was modified with a solvent-exposed cysteine to conjugate monolayer protected gold nanoclusters (AuNC). Unlike commercially available gold nanoparticles, AuNCs monodisperse in water and are composed of a discrete number of gold atoms, forming a crystalline gold core. Au102pMBA44 (Au102) was an ideal conjugate given its small 2.5 nm size and detectability in cryoEM. Au102 was bound directly to nanocapsid surface cysteines via direct ligand exchange. In addition, Au102 was functionalized with a maleimide linker (Au102_C6MI) for maleimide-thiol conjugation to nanocapsid cysteines. The AuNC-bound nanocapsid constructs were conjugated in various conditions. We found Au102_C6MI to bind nanocapsid more efficiently, while Au102 remained more soluble over time. Nanocapsids conjugated to Au102_C6MI were imaged in cryoEM for single particle reconstruction to localize AuNC position on the nanocapsid surface. We resolved five unique high intensity volumes that formed a ring-shaped density at the 5-fold symmetry center. This finding was further supported by independent rigid modeling.
Highlights
Biological constructs with multiple axes of symmetry, such as viruses, are widely used as scaffolds to orient molecules in a uniform arrangement[1,2]
Five cysteine replacement sites were evaluated based on surface exposure, structural flexibility, as well as epitope antigenicity and cell-binding[6]
Based on previous research[6,26] and preliminary screening of AuNC conjugation to various constructs, we selected Hepaptis E Virus (HEV) nanocapsid 573 C to bind AuNCs. 573 C is located on the outer edge of the nanocapsid dimer, just below the upper tip, such that five 573 C sites face the 5-fold symmetry center (Fig. 1b)
Summary
Biological constructs with multiple axes of symmetry, such as viruses, are widely used as scaffolds to orient molecules in a uniform arrangement[1,2]. Chemical conjugation is an efficient method to selectively bind naturally occurring and synthetic molecules to a biological complex. Fluorescently labeled HEV nanocapsids bound to tumor-specific peptides were shown to target tumor tissue in animal models[5,6]. This proof-of-concept study demonstrated the diagnostic potential of chemically modified HEV nanocapsids. Virus-like particles of HEV nanocapsids engineered with a surface-exposed cysteine were bound to molecule-like gold nanoclusters. These AuNC-protein complexes were imaged in EM and structurally characterized with single particle reconstruction. The reconstruction results from this work demonstrated the rigidity of protein-bound AuNCs and suggested larger macromolecules could bind AuNCs functionalized with short linkers
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