Gene Regulation with Polyvalent siRNA−Nanoparticle Conjugates

Abstract

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...

DNA‐Origami‐Driven Lithography for Patterning on Gold Surfaces with Sub‐10 nm Resolution

“…To prepare the conjugates, 1.5 molar excess of thiol-oligonucleotide (DNA-CG: 5'-TGACTCAATGACTCGTTTTTTTTTT-3'-phosphate-(CH2)3-SH), to respect maximum theoretical load of oligonucleotide according to described protocols, [29] . [19] Then, 1/10 of 20 µM OEG-thiol solution in water was added to the OGNP. After incubation with vigorous shaking at RT for 3 hours, the suspension was centrifuged in the same conditions described above.…”

“…[18] Additionally, the resulting OGMPs were passivated with oligoethyleneglycol-thiol [19] to prevent non-specific binding to the gold surface (see Supporting Information).…”

15 years on siRNA delivery: Beyond the State-of-the-Art on inorganic nanoparticles for RNAi therapeutics

“…Later, Mirkin and co-workers reported polyvalent siRNA-AuNP conjugates could be readily taken up by cells and that the particle bound siRNA could effectively regulate genes in the context of RNA interference [24]. In this system, PEG is significantly smaller than the siRNA in order to fully expose the siRNA on the particle surface presented to the cells.…”

“…Gold nanoparticles functionalized with multiple biomolecules: PEG, cell penetration peptide (TAT), ammonium quaternary groups, and siRNA. Two different approaches were employed to conjugate the siRNA to the AuNPs: (A) ionic approach, interaction of the negatively charged siRNA to the modified surface of the AuNPs through ionic interactions; (B) covalent approach, use of thiolated siRNA for gold thiol binding to the In addition, the influence of different parameters such as the thiolation degree of the siRNA [130], the strand involved in the attachment [127], the need of increasing the amount of salt to improve the yield of the functionalization [24] and the possible release of the siRNA with DTT or the glutathione present in the cytosol [38], are also to be taken into account.…”

“…The same is true for studies that use GFP as a reporter gene. As GFP is not a native protein, it has limited used for further studies, which would ideally aim at altering native protein levels by using silencing machinery, such as siRNA, miRNA or small molecules [24,25,38]. When verifying protein knockdown, alternative techniques would need to be used such as western blots [143], flow cytometry [45] and In-Cell westerns [144].…”