We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.
Gold colloids have fascinated scientists for over a century and are now heavily utilized in chemistry, biology, engineering, and medicine. Today these materials can be synthesized reproducibly, modified with seemingly limitless chemical functional groups, and, in certain cases, characterized with atomic-level precision. This Review highlights recent advances in the synthesis, bioconjugation, and cellular uses of gold nanoconjugates. There are now many examples of highly sensitive and selective assays based upon gold nanoconjugates. In recent years, focus has turned to therapeutic possibilities for such materials. Structures which behave as gene-regulating agents, drug carriers, imaging agents, and photoresponsive therapeutics have been developed and studied in the context of cells and many debilitating diseases. These structures are not simply chosen as alternatives to molecule-based systems, but rather for their new physical and chemical properties, which confer substantive advantages in cellular and medical applications.
We demonstrate that novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore-labeled complements can be used as both transfection agents and cellular "nanoflares" for detecting mRNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells.Probes to visualize and detect intracellular RNA including those used for in situ staining, 1 molecular beacons, 2 and fluorescent resonance energy transfer (FRET) pairs 3 are important tools to measure and quantify activity in living systems in response to external stimuli. 4 However, these probes are often difficult to transfect, require additional agents for cellular internalization, and can be unstable in cellular environments. These factors can lead to a high background signal and the inability to detect targets. Here we show how novel oligonucleotide-modified gold nanoparticle probes hybridized to fluorophore complements can be used as both transfection agents and cellular "nano-flares" for visualizing and quantifying RNA in living cells. Nano-flares take advantage of the highly efficient fluorescence quenching properties of gold, 5 cellular uptake of oligonucleotide nanoparticle conjugates without the use of transfection agents, and the enzymatic stability of such conjugates, 6 thus overcoming many of the challenges to creating sensitive and effective intracellular probes. Specifically, nano-flares exhibit high signaling, have low background fluorescence, and are sensitive to changes in the number of RNA transcripts present in cells. The discovery and subsequent development of the oligonucleotide-nanoparticle conjugate have led to a variety of new opportunities in molecular diagnostics 11 and materials design. 12 Recently, it has been demonstrated that oligonucleotide-functionalized nanoparticles enter cells and can act as antisense agents to control gene expression. 6 These "antisense particles" are not only delivery vehicles, 13 but also single entity regulation and transfection agents that undergo facile cellular internalization, resist enzymatic degradation, and bind intracellular targets with affinity constants that are as much as two orders of magnitude greater than free oligonucleotides. 14 Moreover, they can be easily modified with potent designer materials such as locked nucleic acids 15 and are nontoxic under conditions required for gene regulation.The nano-flares described herein are oligonucleotide functionalized nanoparticle conjugates designed to provide an intracellular fluorescence signal that correlates with the relative amount of a specific intracellular RNA. By utilizing nanopart...
The promise of point-of-care medical diagnostics — tests that can be carried out at the site of patient care — is enormous, bringing the benefits of fast and reliable testing and allowing rapid decisions on the course of treatment to be made. To this end, much innovation is occurring in technologies for use in biodiagnostic tests. Assays based on nanomaterials, for example, are now beginning to make the transition from the laboratory to the clinic. But the potential for such assays to become part of routine medical testing depends on many scientific factors, including sensitivity, selectivity and versatility, as well as technological, financial and policy factors.
The cellular internalization of oligonucleotide-modified nanoparticles is investigated. Uptake is dependent on the density of the oligonucleotide loading on the surface of the particles, where higher densities lead to greater uptake. Densely functionalized nanoparticles adsorb a large number of proteins on the nanoparticle surface. Nanoparticle uptake is greatest where a large number of proteins are associated with the particle.
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...
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.
Amine-functionalized polyvalent oligonucleotide gold nanoparticles (DNA-Au NP) were derivatized with a cisplatin prodrug, and the resulting DNA-Au NP conjugates were used to internalize multiple platinum centers. A platinum(IV) complex, c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)], was tethered to the surface of DNA-Au NPs through amide linkages. The platinum-tethered gold nanoparticles (Pt-DNA-Au NPs) were taken into several cancer cells. The drop in intracellular pH facilitated reductive release of cisplatin from the prodrug, which then formed 1,2-d(GpG) intrastrand cross-links in the cell nuclei as confirmed by an antibody specific for this adduct. The cytotoxicity of the platinum(IV) complex increases significantly in several cancer cell lines when the complex is attached to the surface of the DNA-Au NPs and in some instances exceeds that of cisplatin.
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