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 pro-fluorescent, 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. 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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111:3614–3666
Serganov A, Nudler E (2013) A decade of riboswitches. Cell 152:17–24
Gao X et al (2014) Engineered Bacillus subtilis strains allow for rapid characterization of heterologous diguanylate cyclases and phosphodiesterases. Appl Environ Microbiol 80:6167–6174
Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646
Huang H et al (2014) A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol 10:686–691
Warner KD et al (2014) Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat Struct Mol Biol 21:658–663
Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335:1194
Kellenberger CA, Wilson SC, Sales-Lee J, Hammond MC (2013) RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J Am Chem Soc 135:4906–4909
Kellenberger CA, Hammond MC (2015) In vitro analysis of riboswitch-spinach aptamer fusions as metabolite-sensing fluorescent biosensors. Methods Enzymol 550:147–172
Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger cyclic di-GMP: the first 25 years of a universal bacterial. Microbiol Mol Biol Rev 77:1–52
Malone JG et al (2007) The structure-function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain. Microbiology 153:980–994
Novick RP (1987) Plasmid incompatibility. Microbiol Rev 51:381–395
Velappan N, Sblattero D, Chasteen L, Pavlik P, Bradbury ARM (2007) Plasmid incompatibility: more compatible than previously thought? Protein Eng Des Sel 20:309–313
Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4:571–576
Strack RL, Disney MD, Jaffrey SR (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat—containing RNA. Nat Methods 10:1219–1224
Song W, Strack RL, Svensen N, Jaffrey SR (2014) Plug-and-play fluorophores extend the spectral properties of spinach. J Am Chem Soc 136:1198–1201
Acknowledgements
The work on which this chapter is based was supported by NIH grant DP2 OD008677 (to M.C.H.), DoD NDSEG fellowship (to C.A.K.) and NSF graduate fellowship (to Z.F.H.). M.C.H. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. The authors thank Xin Cindy Wang for providing feedback on manuscript edits.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kellenberger, C.A., Hallberg, Z.F., Hammond, M.C. (2015). Live Cell Imaging Using Riboswitch-Spinach tRNA Fusions as Metabolite-Sensing Fluorescent Biosensors. In: Ponchon, L. (eds) RNA Scaffolds. Methods in Molecular Biology, vol 1316. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2730-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2730-2_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2729-6
Online ISBN: 978-1-4939-2730-2
eBook Packages: Springer Protocols