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Live Cell Imaging Using Riboswitch–Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors

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RNA Scaffolds

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2323))

Abstract

The development of fluorescent biosensors is motivated by the desire to monitor cellular metabolite levels in real time. Most genetically encodable fluorescent biosensors are based on receptor proteins fused to fluorescent protein domains. More recently, small molecule–binding riboswitches have been adapted for use as fluorescent biosensors through fusion to the in vitro selected Spinach aptamer, which binds a profluorescent, cell-permeable small molecule mimic of the GFP chromophore, DFHBI. Here we describe methods to prepare and analyze riboswitch–Spinach tRNA fusions for ligand-dependent activation of fluorescence in vivo. Example procedures describe the use of the Vc2-Spinach tRNA biosensor to monitor perturbations in cellular levels of cyclic di-GMP using either fluorescence microscopy or flow cytometry. In this updated chapter, we have added procedures on using biosensors in flow cytometry to detect exogenously added compounds. The relative ease of cloning and imaging of these biosensors, as well as their modular nature, should make this method appealing to other researchers interested in utilizing riboswitch-based biosensors for metabolite sensing.

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Acknowledgments

The work on which this updated chapter is based was supported by Office of Naval Research grant N000141712638 (to M.C.H.).

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Authors and Affiliations

  1. Department of Chemistry, University of Utah, Salt Lake City, UT, USA

    Sudeshna Manna & Ming C. Hammond

  2. Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, UT, USA

    Sudeshna Manna & Ming C. Hammond

  3. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Colleen A. Kellenberger, Zachary F. Hallberg & Ming C. Hammond

Authors
  1. Sudeshna Manna
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  2. Colleen A. Kellenberger
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  3. Zachary F. Hallberg
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  4. Ming C. Hammond
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Corresponding author

Correspondence to Ming C. Hammond .

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Editors and Affiliations

  1. Faculté de Pharmacie, University of Paris, PARIS, France

    Luc Ponchon

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Manna, S., Kellenberger, C.A., Hallberg, Z.F., Hammond, M.C. (2021). Live Cell Imaging Using Riboswitch–Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors. In: Ponchon, L. (eds) RNA Scaffolds. Methods in Molecular Biology, vol 2323. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1499-0_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1499-0_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1498-3

  • Online ISBN: 978-1-0716-1499-0

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