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Author Patel, Pinal Chandrakant
Title Emergent properties of oligonucleotide-gold nanoparticle conjugates
book jacket
Descript 183 p
Note Source: Dissertation Abstracts International, Volume: 72-08, Section: B, page:
Adviser: Chad A. Mirkin
Thesis (Ph.D.)--Northwestern University, 2011
Polyvalent oligonucleotide-gold nanoparticle conjugates are a class of hybrid materials that consist of an inorganic core functionalized with a dense monolayer of synthetic oligonucleotides. Due to the dense arrangement of oligonucleotides, they exhibit resistance to nuclease degradation, enhanced binding to complementary oligonucleotide targets, and autonomous and efficient cellular uptake. These emergent properties make them an exciting tool to study cellular biology and mediate oligonucleotide directed therapeutic work
In Chapter 2, a novel heterofunctionalized nanoparticle conjugate consisting of a 13 nm gold nanoparticle (Au NP) containing both antisense oligonucleotides and synthetic peptides is described. The synthesis of this conjugate was accomplished by mixing thiolated oligonucleotides and cysteine-terminated peptides with gold nanoparticles in the presence of salt which screens interactions between biomolecules yielding a densely functionalized nanomaterial. By controlling the stoichiometry of the components in solution, one can control the surface loading of each biomolecule. The conjugates are easily prepared, show perinuclear localization and an enhanced gene regulation activity when tested in a cellular model. This heterofunctionalized structure represents a new strategy for preparing nanomaterials with potential therapeutic applications
In Chapter 3, Structural requirements of siRNA-functionalized gold nanoparticles (siRNA-Au NPs) for Dicer recognition and serum stability are studied. It is shown that in the context of siRNA-Au NPs, the 3' overhang is preferentially recognized by Dicer but also makes the siRNA duplexes more susceptible to non-specific serum degradation. Dicer and serum nucleases show lower preference for blunt duplexes as opposed to those with 3' overhangs. Importantly, gold nanoparticles functionalized with blunt duplexes with low thermal breathing are up to 15 times more stable against serum degradation without compromising Dicer recognition. This increased stability leads to 3-fold increase in cellular uptake of siRNA-Au NPs and improved gene knockdown
In Chapter 4, the mechanism of uptake of polyvalent nucleic acids nanostructures is elucidated. Mammalian cells have been shown to internalize oligonucleotide-functionalized gold nanoparticles (DNA-Au NPs or siRNA-Au NPs) without the aid of auxiliary transfection agents and use them to initiate an antisense or RNAi response. Previous studies have shown that the dense monolayer of oligonucleotides on the nanoparticle leads to the adsorption of serum proteins and facilitates cellular uptake. It is observed that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. The pathway for DNA-Au NPs entry in HeLa cells is identified. Biochemical analyses indicate that DNA-Au NPs are taken up by a process involving receptor-mediated endocytosis. Evidence shows that DNA-Au NPs entry is primarily mediated by scavenger receptors, a class of pattern-recognition receptors. This uptake mechanism appears to be conserved across species as blocking the same receptors in mouse cells also disrupted DNA-Au NP entry. Polyvalent nanoparticles functionalized with siRNA are shown to enter through the same pathway. Thus, scavenger receptors are required for cellular uptake of polyvalent oligonucleotide functionalized nanoparticles
In Chapter 5, we used the properties discovered in previous chapters for the synthesis and characterization of mimic microRNA-gold nanoparticle conjugates (mimic miRNA-Au NPs), nanoparticles that are densely functionalized with synthetic mimic miRNA oligonucleotides and designed to function analogously to endogenous miRNAs. We show that the nanoparticles carrying the mimics of tumor suppressive microRNA miR-205 repress the expression of miR-205 target protein by interaction with the 3' untranslated regions (3'-UTR) of the target mRNA. We further demonstrate that the mimic miRNA-Au NPs exert inhibition of tumor cell proliferation in human prostate cancer cell lines. By contrast, the nanoparticles functionalized with mimics of oncogenic miR-20a promote cell survival by down-regulating known miR-20a target proteins. These polyvalent structures are introduced without the use of a toxic co-carrier, and overcome several challenges related to miRNA introduction. Furthermore, these constructs regulate gene expression up to three fold more effectively than analogous molecular systems. Thus, nucleic acid-nanoparticle conjugates which function as mimics of microRNAs provide a novel tool for studying miRNA function as well as potential miRNA replacement therapy
School code: 0163
Host Item Dissertation Abstracts International 72-08B
Subject Biology, Cell
Chemistry, Biochemistry
Nanotechnology
0379
0487
0652
Alt Author Northwestern University. Interdepartmental Biological Sciences Program
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