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      Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach

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          Abstract

          Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of ( Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1 H-imidazol-5(4 H)-one (DFHBI), allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Here we characterize the structural features that are required for fluorophore binding to Spinach2 and describe novel fluorophores that bind and are switched to a fluorescent state by Spinach2. These diverse Spinach2–fluorophore complexes exhibit fluorescence that is more compatible with existing microscopy filter sets and allows Spinach2-tagged constructs to be imaged with either GFP or YFP filter cubes. Thus, these “plug-and-play” fluorophores allow the spectral properties of Spinach2 to be altered on the basis of the specific spectral needs of the experiment.

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          Most cited references15

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          Rapid planetesimal formation in turbulent circumstellar discs

          The initial stages of planet formation in circumstellar gas discs proceed via dust grains that collide and build up larger and larger bodies (Safronov 1969). How this process continues from metre-sized boulders to kilometre-scale planetesimals is a major unsolved problem (Dominik et al. 2007): boulders stick together poorly (Benz 2000), and spiral into the protostar in a few hundred orbits due to a head wind from the slower rotating gas (Weidenschilling 1977). Gravitational collapse of the solid component has been suggested to overcome this barrier (Safronov 1969, Goldreich & Ward 1973, Youdin & Shu 2002). Even low levels of turbulence, however, inhibit sedimentation of solids to a sufficiently dense midplane layer (Weidenschilling & Cuzzi 1993, Dominik et al. 2007), but turbulence must be present to explain observed gas accretion in protostellar discs (Hartmann 1998). Here we report the discovery of efficient gravitational collapse of boulders in locally overdense regions in the midplane. The boulders concentrate initially in transient high pressures in the turbulent gas (Johansen, Klahr, & Henning 2006), and these concentrations are augmented a further order of magnitude by a streaming instability (Youdin & Goodman 2005, Johansen, Henning, & Klahr 2006, Johansen & Youdin 2007) driven by the relative flow of gas and solids. We find that gravitationally bound clusters form with masses comparable to dwarf planets and containing a distribution of boulder sizes. Gravitational collapse happens much faster than radial drift, offering a possible path to planetesimal formation in accreting circumstellar discs.
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            Impaired respiratory and body temperature control upon acute serotonergic neuron inhibition.

            Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved, but their constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a neuronal silencing tool, mouse RC::FPDi (based on the synthetic G protein-coupled receptor Di), designed for cell type-specific, ligand-inducible, and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, administration of the ligand clozapine-N-oxide (CNO) by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue carbon dioxide (CO(2)) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO(2) acidosis. Body thermoregulation at room temperature was also disrupted after CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.
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              A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat RNA

              Fluorescent imaging of RNA in living cells is a technically challenging problem in cell biology. One strategy for genetically encoding fluorescent RNAs is to express them as fusions with ‘RNA mimics of GFP’. These are short aptamer tags that exhibit fluorescence upon binding otherwise nonfluorescent fluorophores that resemble those found in GFP. We find that the brightest of these aptamers, Spinach, often exhibits reduced fluorescence after it is fused to RNAs of interest. We show that a combination of thermal instability and a propensity for misfolding account for the low fluorescence of various Spinach-RNA fusions. Using systematic mutagenesis, we identified nucleotides that account for the poor folding of Spinach, and generated Spinach2, which exhibits markedly improved thermal stability and folding in cells. Furthermore, we show that Spinach2 largely retains its fluorescence when fused to various RNAs. Using Spinach2, we detail the cellular dynamics of the CGG trinucleotide-repeat containing “toxic RNA” associated with Fragile-X tremor/ataxia syndrome, and show that these RNAs form nuclear foci with unexpected morphological plasticity that is regulated by the cell cycle and by small molecules. Together, these data demonstrate that Spinach2 exhibits improved versatility for fluorescently labeling RNAs in living cells.
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                Author and article information

                Journal
                J Am Chem Soc
                J. Am. Chem. Soc
                ja
                jacsat
                Journal of the American Chemical Society
                American Chemical Society
                0002-7863
                1520-5126
                06 January 2014
                29 January 2014
                : 136
                : 4
                : 1198-1201
                Affiliations
                [1]Department of Pharmacology, Weill Medical College, Cornell University , New York, New York 10065, United States
                Author notes
                Article
                10.1021/ja410819x
                3929357
                24393009
                0dab1b6d-1631-4a43-8b38-9bc00100b2a4
                Copyright © 2014 American Chemical Society
                History
                : 22 October 2013
                Funding
                National Institutes of Health, United States
                Categories
                Communication
                Custom metadata
                ja410819x
                ja-2013-10819x

                Chemistry
                Chemistry

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