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...
Nanoparticles from colloidal solutionwith controlled composition, size, and shapeserve as excellent building blocks for plasmonic devices and metasurfaces. However, understanding hierarchical driving forces affecting the geometry of oligomers and interparticle gap spacings is still needed to fabricate high-density architectures over large areas. Here, electrohydrodynamic (EHD) flow is used as a long-range driving force to enable carbodiimide cross-linking between nanospheres and produces oligomers exhibiting sub-nanometer gap spacing over mm 2 areas. Anhydride linkers between nanospheres are observed via surface-enhanced Raman scattering (SERS) spectroscopy. The anhydride linkers are cleavable via nucleophilic substitution and enable placement of nucleophilic molecules in electromagnetic hotspots. Atomistic simulations elucidate that the transient attractive force provided by EHD flow is needed to provide a sufficient residence time for anhydride cross-linking to overcome slow reaction kinetics. This synergistic analysis shows assembly involves an interplay between long-range driving forces increasing nanoparticle− nanoparticle interactions and probability that ligands are in proximity to overcome activation energy barriers associated with short-range chemical reactions. Absorption spectroscopy and electromagnetic full-wave simulations show that variations in nanogap spacing have a greater influence on optical response than variations in close-packed oligomer geometry. The EHD flow−anhydride cross-linking assembly method enables close-packed oligomers with uniform gap spacings that produce uniform SERS enhancement factors. These results demonstrate the efficacy of colloidal driving forces to selectively enable chemical reactions leading to future assembly platforms for large-area nanodevices.
We use frequency-modulated electrostatic force microscopy to track changes in cantilever quality factor (Q) as a function of photochemical damage in a model organic photovoltaic system poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl]] (PTB7) and 3'H-cyclopropa[8,25][5,6]fullerene-C71-D5h(6)-3'-butanoic acid, 3'-phenyl-, methyl ester (PC71BM). We correlate local Q factor imaging with macroscopic device performance and show that, for this system, changes in cantilever Q correlate well with changes in external quantum efficiency and can thus be used to monitor local photochemical damage over the entire functional lifetime of a PTB7:PC71BM solar cell. We explore how Q imaging is affected by the choice of cantilever resonance frequency. Finally, we use Q imaging to elucidate the differences in the evolution of nanoscale structure in the photochemical damage occurring in PTB7:PC71BM solar cells processed with and without the solvent additive 1,8-diiodooctane (DIO). We show that processing with DIO not only yields a preferable morphology for uniform performance across the surface of the device but also enhances the stability of PTB7:PC71BM solar cells-an effect that can be predicted based on the local Q images.
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.
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.
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