Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymatic degradation. Stability in the presence of enzymes is a key consideration for therapeutic uses; however the manner and mechanism by which such nanoparticles are able to resist enzymatic degradation is unknown. Here, we quantify the enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates with respect to enzyme-catalyzed hydrolysis of DNA and present evidence that the negatively charged surfaces of the nanoparticles and resultant high local salt concentrations are responsible for enhanced stability.

We report the synthesis and characterization of polyvalent RNA-gold nanoparticle conjugates (RNAAu NPs), nanoparticles that are densely functionalized with synthetic RNA oligonucleotides and designed to function in the RNAi pathway. The particles were rationally designed and synthesized to be free of degrading enzymes, have a high surface loading of siRNA duplexes, and contain an auxiliary passivating agent for increased stability in biological media. The resultant conjugates have a half-life six times longer than free dsRNA, readily enter cells without the use of transfection agents, and demonstrate a high gene knockdown capability in a cell model.Over the past decade, researchers have designed, synthesized, studied, and applied polyvalent DNA-functionalized gold nanoparticles (DNA-Au NPs). 1 These efforts have resulted in a new fundamental understanding of hybrid nanostructures,2 important and in certain cases commercially viable detection and diagnostic assays,3 and the ability to program materials assembly through the use of DNA synthons.1 , 4 Polyvalent DNA-Au NPs have several unique properties, such as sharp and elevated melting temperatures,2b enhanced binding properties 2c (as compared with free strands of the same sequence), and distance-dependent optical properties. 5 In agreement with research on polyvalent molecular systems,6 the high surface DNA density and the ability of the nanoparticles to engage in multidentate interactions are the proposed origin of these unique properties.Recently, we demonstrated the utility of the polyvalent DNA-Au NP for antisense gene regulation, where the unique ensemble properties of the conjugate confer several important advantages in the context of intracellular target recognition and binding. 7 These properties include resistance to nuclease degradation and high cellular uptake as a result of their oligonucleotide functionalization. Although antisense DNA is an important way of regulating genes, an even more promising route is through the use of siRNA. 8 However, no methods have been developed for utilizing polyvalent particles and their unusual properties to load and transport RNA across cell membranes. Indeed, one must develop synthetic routes and materials that overcome one of the most challenging problems associated with RNA, most notably its chemical instability.Based upon our observations with DNA-modified particles, we hypothesized that gold nanoparticles densely functionalized with synthetic RNA oligonucleotides would take advantage of the ensemble properties that result from the dense surface functionalization of oligonucleotides, increase the stability and efficacy of the bound RNA, while retaining the ability to act in the highly potent and catalytic RNA interference pathway. While others have chadnano@northwestern.edu. Supporting Information Available: Experimental conditions, sequences, and materials synthesized. This material is available free of charge via the Internet at http://pubs.acs.org. We determined that treatment of citrate-capped Au NPs...

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. Here, we show that serum proteins generally act to inhibit cellular uptake of DNA-Au NPs. We identify the pathway for DNA-Au NPs entry in HeLa cells. 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.

We build off the previously described concept of a nano-flare to develop an oligonucleotide gold nanoparticle conjugate that is capable of both detecting and regulating intracellular levels of mRNA. We characterize the binding rate and specificity of these materials using survivin, a gene associated with the diagnosis and treatment of cancer, as a target. The nanoconjugate enters cells and binds mRNA, thereby decreasing the relative abundance of mRNA in a dose-and sequence-dependent manner and resulting in a fluorescent response. This represents the first demonstration of a single material capable of both mRNA regulation and detection. Further, we investigate the intracellular biochemistry of the nanoconjugate, elucidating its mechanism of gene regulation. This work is important to the study of biologically active nanomaterials such as the nano-flare and is a first step towards the development of an mRNA responsive 'theranostic'. KeywordsNanoparticle; oligonucleotide; mRNA; detection; gene regulation; theranostic Over the past decade, researchers have investigated the conjugation of biomolecules to inorganic nanomaterials, which has led to the development of hybrid materials with new activities. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] One important class of hybrid nanomaterial is composed of a gold nanoparticle functionalized with a dense monolayer of oligonucleotides. These polyvalent nanoconjugates have many interesting properties, including distance-dependent optical features, 21,22 enhanced nucleic acid binding, 23 resistance to degradation, 24 and the ability to enter cells without use of transfection agents. 25 These remarkable properties have enabled controlled assembly of materials, 26-28 molecular diagnostics, [29][30][31][32] and intracellular studies. [33][34][35] Materials with both regulation and detection capabilities are of growing interest for use in personalized medicine. 36 These 'theranostic' materials have the potential to both treat and diagnose disease and are useful for investigating intracellular events (i.e. target recognition and control of biological function). Traditionally, antisense oligonucleotides and molecular beacons have been used to regulate and detect intracellular mRNA, respectively. Antisense oligonucleotides regulate gene expression by binding target mRNA and preventing translation. 37 Molecular beacons detect nucleic acids by coupling a binding event with a signal transduction mechanism, such as the separation of a fluorophore-quencher pair. 38 Given that cell entry and mRNA binding are the first steps in both processes, it should be possible to design a single material for both regulation and detection. To the best of our knowledge, however, a material capable of both mRNA regulation and detection has not been reported. 39 Such materials must be readily taken up by cells, stable in intracellular environments, capable of binding nucleic acids, and possess a switchable signal that can be conveniently detected. NIH Public Access RES...

We report the development of the multiplexed nano-flare, a nanoparticle agent that is capable of simultaneously detecting two distinct messenger RNA (mRNA) targets inside a living cell. These probes consist of polyvalent DNA-functionalized gold nanoparticles with multiple DNA sequences, each hybridized to a reporter with a distinct fluorophore label, and each complementary to its corresponding mRNA target. When multiplexed nano-flares are exposed to their targets, they provide a sequence specific signal in both extra- and intracellular environments. Importantly, one of the targets can be used as an internal control, improving detection by accounting for cell-to-cell variations in nanoparticle uptake and background. Compared to single-component nano-flares, these structures allow one to determine more precisely relative mRNA levels in individual cells, improving cell sorting and quantification.

Polyvalent oligonucleotide-nanoparticle conjugates possess several unique emergent properties including enhanced cellular uptake, high antisense bioactivity, and nuclease resistance, which hypothetically originate from the dense packing and orientation of oligonucleotides on the surface of the nanoparticle. In this communication, we describe a new class of polyvalent nucleic acid nanostructures (PNANs), which comprise only crosslinked and oriented nucleic acids. We demonstrate that these particles are capable of effecting high cellular uptake and gene regulation without the need of a cationic polymer co-carrier. The PNANs also exhibit cooperative binding behavior and nuclease resistance properties.

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We demonstrate that polyvalent DNA-functionalized gold nanoparticles (DNA-Au NPs) selectively enhance Ribonuclease H (RNase H) activity, while inhibiting most biologically relevant nucleases. This combination of properties is particularly interesting in the context of gene regulation, since high RNase H activity results in rapid mRNA degradation and general nuclease inhibition results in high biological stability. We investigate the mechanism of selective RNase H activation and find that the high DNA density of DNA-Au NPs is responsible for this unusual behavior. This work adds to our understanding of polyvalent DNA-Au NPs as gene regulation agents, and suggests a new model for selectively controlling protein-nanoparticle interactions.