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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Newman RH, Fosbrink MD, Zhang J (2011) Genetically Encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111(5):3614–3666
Serganov A, Nudler E (2013) A decade of riboswitches. Cell 152(1-2):17–24
Gao X, Dong X, Subramanian S, Matthews PM, Cooper CA, Kearns DB, Dann CE 3rd (2014) Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases. Appl Environl Microbiol 80(19):6167–6174
Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333(6042):642–646
Huang H, Suslov NB, Li N-S, Shelke SA, Evans ME, Koldobskaya Y, Rice PA, Piccirilli JA (2014) A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore. Nat Chem Biol 10(8):686–691
Warner KD, Chen MC, Song W, Strack RL, Thorn A, Jaffrey SR, Ferré-D'Amaré AR (2014) Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nat Struct Mol Biol 21(8):658–663
Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335(6073):1194–1194
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(13):4906–4909
Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77(1):1–52
Strack RL, Disney MD, Jaffrey SR (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat–containing RNA. Nat Methods 10(12):1219–1224
Wang XC, Wilson SC, Hammond MC (2016) Next-generation RNA-based fluorescent biosensors enable anaerobic detection of cyclic di-GMP. Nucleic Acids Res 44(17):e139
Yeo J, Dippel AB, Wang XC, Hammond MC (2018) In vivo biochemistry: single-cell dynamics of cyclic Di-GMP in Escherichia coli in response to zinc overload. Biochemistry 57(1):108–116
Kellenberger CA, Hallberg ZF, Hammond MC (2015) Live cell imaging using riboswitch-spinach tRNA fusions as metabolite-sensing fluorescent biosensors. In: Ponchon L (ed) RNA scaffolds: methods and protocols. Springer New York, New York, NY, pp 87–103
Manna S, Truong J, Hammond MC (2021) Guanidine biosensors enable comparison of cellular turn-on kinetics of riboswitch-based biosensor and reporter. ACS Synth Biol 10(3):566–578
Stojanovic MN, Kolpashchikov DM (2004) Modular aptameric sensors. J Am Chem Soc 126(30):9266–9270
Furutani C, Shinomiya K, Aoyama Y, Yamada K, Sando S (2010) Modular blue fluorescent RNA sensors for label-free detection of target molecules. Mol BioSyst 6(9):1569–1571
Kellenberger CA, Wilson SC, Hickey SF, Gonzalez TL, Su Y, Hallberg ZF, Brewer TF, Iavarone AT, Carlson HK, Hsieh Y-F, Hammond MC (2015) GEMM-I riboswitches fromGeobacter sense the bacterial second messenger cyclic AMP-GMP. Proc Natl Acad Sci U S A 112(17):5383
Kellenberger CA, Chen C, Whiteley AT, Portnoy DA, Hammond MC (2015) RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J Am Chem Soc 137(20):6432–6435
You M, Litke JL, Jaffrey SR (2015) Imaging metabolite dynamics in living cells using a spinach-based riboswitch. Proc Natl Acad Sci U S A 112(21):E2756
Su Y, Hickey SF, Keyser SGL, Hammond MC (2016) In vitro and in vivo enzyme activity screening via RNA-based fluorescent biosensors for S-Adenosyl-l-homocysteine (SAH). J Am Chem Soc 138(22):7040–7047
Bose D, Su Y, Marcus A, Raulet DH, Hammond MC (2016) An RNA-based fluorescent biosensor for high-throughput analysis of the cGAS-cGAMP-STING pathway. Cell Chem Biol 23(12):1539–1549
Porter EB, Polaski JT, Morck MM, Batey RT (2017) Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors. Nat Chem Biol 13(3):295–301
Jepsen MDE, Sparvath SM, Nielsen TB, Langvad AH, Grossi G, Gothelf KV, Andersen ES (2018) Development of a genetically encodable FRET system using fluorescent RNA aptamers. Nat Commun 9(1):18
You M, Litke JL, Wu R, Jaffrey SR (2019) Detection of low-abundance metabolites in live cells using an RNA integrator. Cell Chem Biol 26(4):471–481
Wu R, Karunanayake Mudiyanselage APKK, Ren K, Sun Z, Tian Q, Zhao B, Bagheri Y, Lutati D, Keshri P, You M (2020) Ratiometric Fluorogenic RNA-based sensors for imaging live-cell dynamics of small molecules. ACS Appl. Bio Mater 5:2633–2642. https://doi.org/10.1021/acsabm.9b01237
Su Y, Hammond MC (2020) RNA-based fluorescent biosensors for live cell imaging of small molecules and RNAs. Curr Opin Biotec 63:157–166
Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expres Purif 41(1):207–234
Song W, Strack RL, Svensen N, Jaffrey SR (2014) Plug-and-play fluorophores extend the spectral properties of spinach. J Am Chem Soc 136(4):1198–1201
Malone JG, Williams R, Christen M, Jenal U, Spiers AJ, Rainey PB (2007) The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain. Microbiology 153(4):980–994
Novick RP (1987) Plasmid incompatibility. Microbiol Rev 51(4):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(7):309–313
Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4(7):571–576
Acknowledgments
The work on which this updated chapter is based was supported by Office of Naval Research grant N000141712638 (to M.C.H.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1499-0_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1498-3
Online ISBN: 978-1-0716-1499-0
eBook Packages: Springer Protocols