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.

Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nmdiameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo-and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.f 27 million Americans diagnosed with type 2 diabetes (T2D), more than 6 million have chronic, nonhealing skin wounds, particularly on the plantar surface, leading to secondary bacterial infection and costing the healthcare system more than $25 billion (1, 2). In 2010 alone, more than 70,000 individuals in the United States with T2D underwent amputation (3). Improved understanding of diabetic wound pathology and new interventions for impaired wound healing are needed.Ganglioside-monosialic acid 3 (GM3), the predominant sialylated glycosphingolipid in skin, has recently been recognized to be a critical mediator of insulin resistance (4-12). Indeed, we have recently shown three-and fourfold more GM3 synthase (GM3S; also known as SAT-I or ST3Gal-V), which is required for the synthesis of GM3, in diabetic human plantar skin than in site-and age-matched control skin (4). Similarly, skin samples from the backs of diet-induced obese (DIO) and ob/ob mouse diabetic models show increased GM3S mRNA expression and GM3 levels. Knockout (KO) of GM3S improves the insulin resistance induced by a high-fat diet in mouse adipose tissue, muscle (5), and as recently shown, skin of DIO T2D mice, reversing the woundhealing impairment of T2D (4). The acceleration of wound healing by GM3S KO and GM3 depletion in mouse skin is associated with increased epidermal cell migration and proliferation as well as activation of the epidermal insulin-like growth factor-1 receptor (IGF1R) in vivo (4). Isolated cultured mouse GM3S −/− keratinocytes (KCs) migrate and proliferate more rapidly than GM3S +/+ WT l...

We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.he study of RNA is a critical component of biological research and in the diagnosis and treatment of disease. Recently, the localization of mRNA has been identified as an essential process for a number of cellular functions, including restricting the production of certain proteins to specific compartments within cells (1). For instance, synaptic potentiation, the basis of learning and memory, relies on the local translation of specific mRNAs in pre-and postsynaptic compartments (2). Likewise, the misregulation of RNA distribution is associated with many disorders, including mental retardation, autism, and cancer metastasis (3-5). However, despite the significant role of mRNA transport and localization in cellular function, the available methods to visualize these phenomena are severely limited. For example, FISH, the most commonly used technique to analyze spatial distribution of RNA, requires fixation and permeabilization of cells before analysis (6). As a result, analysis of dynamic RNA distribution is restricted to a single snapshot in time (7,8). With such a limitation, understanding the translocation of RNA with respect to time, cell cycle, or external stimulus is difficult if not impossible. Furthermore, fixed cell analysis is a lengthy and highly specialized procedure due to the number of steps necessary to prepare a sample. Fixation, permeabilization, blocking, and staining processes each require optimization and vary based on cell type and treatment conditions, rendering FISH prohibitively complicated in many cases. Likewise, live cell analysis platforms such as molecular beacons require toxic transfection techniques, such as microinjection or lipid transfection, and are rapidly sequestered to the nucleus on cellular entry (9, 10). Recently more sophisticated live cell analyses have been developed that use genetic engineering to introduce exogenous hybrid gene...

The hybridization of free oligonucleotides to densely packed, oriented arrays of DNA modifying the surfaces of spherical nucleic acid (SNA)–gold nanoparticle conjugates occurs with negative cooperativity; i.e., each binding event destabilizes subsequent binding events. DNA hybridization is thus an ever-changing function of the number of strands already hybridized to the particle. Thermodynamic quantification of this behavior reveals a 3 orders of magnitude decrease in the binding constant for the capture of a free oligonucleotide by an SNA conjugate as the fraction of pre-hybridized strands increases from 0 to ∼30%. Increasing the number of pre-hybridized strands imparts an increasing enthalpic penalty to hybridization that makes binding more difficult, while simultaneously decreasing the entropic penalty to hybridization, which makes binding more favorable. Hybridization of free DNA to an SNA is thus governed by both an electrostatic barrier as the SNA accumulates charge with additional binding events and an effect consistent with allostery, where hybridization at certain sites on an SNA modify the binding affinity at a distal site through conformational changes to the remaining single strands. Leveraging these insights allows for the design of conjugates that hybridize free strands with significantly higher efficiencies, some of which approach 100%.

Herein, we report the synthesis of DNA-functionalized infinite coordination polymer (ICP) nanoparticles as biocompatible gene regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of FeIII to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the FeIII-HOPO ICP particles, via copper-free click chemistry, afforded colloidally stable nucleic acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibit narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.