Browsing by Author "Dr.Stefan Franzen, Committee Chair"

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    Development of Red clover necrotic mosaic virus as a Multifunctional Nanoparticle
    (2009-01-02) Loo, Li Na; Dr.Stefan Franzen, Committee Chair
    Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and the family Tombusviridae. RCNMV is a plant virus with 180 identical capsid proteins (CP) arranged to form a T = 3 icosahedral symmetry. The capsid, with an outer diameter of 36 nm and an inner diameter of 17 nm, is enclosed to package two ssRNA: RNA-1 (3.9 kb) and RNA-2 (1.5 kb). In nature, a 20 nucleotide hairpin structure within the RNA-2 sequence trans-activator region forms a kissing loop complex with 8 nucleotides of the RNA-1 to serve as the origin of assembly (OAS). This pseudo-knot structure selectively recruits CP to initiate the virus assembly. In one of the research directions, an oligonucleotide mimic of the OAS sequence was attached to Au, CoFe2O4, and CdSe nanoparticles ranging from 3-15 nm, to trigger virus-like assembly by RCNMV CP. The formation of virus-like particles encapsidated nanoparticles was comparable in size to the native RCNMV. Attempts to encapsidate nanoparticles with diameters > 17 nm did not result in well-formed particles. The versatility of RCNMV capsid to package smaller cargo was explored. RCNMV experiences reversible swelling transition in a pH- and divalent ion-dependent manner. This transition leads to the reversible opening of pores (~13 Å) that permit infusion of molecules into the interior cavity. Comparison of three dyes, Rhodamine (positive charge), Luminarosine (neutral) and Fluorescein (negative charge) shows that infusion is controlled by electrostatic interactions. This method was applied to package an anticancer drug, Doxorubicin, within the capsid. The capability of the RCNMV capsid to deliver cargo into cancer cells was explored. Peptides from various serotypes of adenovirus were conjugated to the capsid to determine the internalization efficiency. The modified RCNMV was prepared with packaged molecules in the interior cavity and targeting peptide on the exterior surface. Intracellular uptake was studied both in terms of RCNMV internalization and cargo release. The utilization of RCNMV capsid as a multifunctional tool for therapeutic purposes demonstrates a new general therapeutic and diagnostic platform.