Fluorescence-Based Methods for Characterizing RNA Interactions In Vivo

  • Abigail N. Leistra
  • Mia K. Mihailovic
  • Lydia M. ContrerasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1737)


Fluorescence-based tools that measure RNA-RNA and RNA-protein interactions in vivo offer useful experimental approaches to probe the complex and dynamic physiological behavior of bacterial RNAs. Here we document the step-by-step design and application of two fluorescence-based methods for studying the regulatory interactions RNAs perform in vivo: (i) the in vivo RNA Structural Sensing System (iRS3) for measuring RNA accessibility and (ii) the trifluorescence complementation (TriFC) assay for measuring RNA-protein interactions.


RNA-RNA interaction RNA-protein interaction In vivo fluorescence assay Hybridization efficacy RNA accessibility Complementation assay RNA regulator Protein regulator Target network 



This work is supported by the Welch Foundation (Grant F-1756 to L.M.C.), and the National Science Foundation (Grant MCB 1716777 to L.M.C., and DGE-1610403 to A.N.L. and M.K .M.).


  1. 1.
    Vazquez-Anderson J, Contreras LM (2013) Regulatory RNAs: charming gene management styles for synthetic biology applications. RNA Biol 10(12):1778–1797. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Tsai C-H, Liao R, Chou B, Palumbo M, Contreras LM (2015) Genome-wide analyses in bacteria show small-RNA enrichment for long and conserved intergenic regions. J Bacteriol 197(1):40–50. CrossRefPubMedGoogle Scholar
  3. 3.
    Cho SH, Lei R, Henninger TD, Contreras LM (2014) Discovery of ethanol responsive small RNAs in Zymomonas mobilis. Appl Environ Microbiol 80(14):4189–4198. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jones AJ, Venkataramanan KP, Papoutsakis T (2016) Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum. FEMS Microbiol Lett 363(8):fnw063. CrossRefPubMedGoogle Scholar
  5. 5.
    Sowa SW, Vazquez-Anderson J, Clark CA, De La Peña R, Dunn K, Fung EK, Khoury MJ, Contreras LM (2015) Exploiting post-transcriptional regulation to probe RNA structures in vivo via fluorescence. Nucleic Acids Res 43(2):e13. CrossRefPubMedGoogle Scholar
  6. 6.
    Watters KE, Abbott TR, Lucks JB (2015) Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq. Nucleic Acids Res 44(2):e12. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ignatova Z, Narberhaus F (2017) Systematic probing of the bacterial RNA structurome to reveal new functions. Curr Opin Microbiol 36:14–19. CrossRefPubMedGoogle Scholar
  8. 8.
    Strobel EJ, Watters KE, Loughrey D, Lucks JB (2016) RNA systems biology: uniting functional discoveries and structural tools to understand global roles of RNAs. Curr Opin Biotechnol 39:182–191. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Garst AD, Edwards AL, Batey RT (2011) Riboswitches: structures and mechanisms. Cold Spring Harb Perspect Biol 3(6):a003533. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Takahashi MK, Watters KE, Gasper PM, Abbott TR, Carlson PD, Chen AA, Lucks JB (2016) Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators. RNA 22(6):920–933. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Vazquez-Anderson J, Mihailovic MK, Baldridge KC, Reyes KG, Haning K, Cho SH, Amador P, Powell WB, Contreras LM (2017) Optimization of a novel biophysical model using large scale in vivo antisense hybridization data displays improved prediction capabilities of structurally accessible RNA regions. Nucleic Acids Res 45(9):5523–5538. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lim F, Peabody DS (1994) Mutations that increase the affinity of a translational repressor for RNA. Nucleic Acids Res 22(18):3748–3752. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    C-D H, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9(4):789–798. CrossRefGoogle Scholar
  14. 14.
    Gelderman G, Sivakumar A, Lipp S, Contreras LM (2015) Adaptation of tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria. Biotechnol Bioeng 112(2):365–375. CrossRefPubMedGoogle Scholar
  15. 15.
    Sowa SW, Gelderman G, Leistra AN, Buvanendiran A, Lipp S, Pitaktong A, Vakulskas CA, Romeo T, Baldea M, Contreras LM (2017) Integrative FourD omics approach profiles the target network of the carbon storage regulatory system. Nucleic Acids Res 45(4):1673–1686. PubMedPubMedCentralGoogle Scholar
  16. 16.
    Gripenland J, Netterling S, Loh E, Tiensuu T, Toledo-Arana A, Johansson J (2010) RNAs: regulators of bacterial virulence. Nat Rev Microbiol 8(12):857–866CrossRefPubMedGoogle Scholar
  17. 17.
    Storz G, Vogel J, Wassarman Karen M (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43(6):880–891. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. CrossRefPubMedGoogle Scholar
  19. 19.
    Green AA, Silver PA, Collins JJ, Yin P (2014) Toehold switches: de-novo-designed regulators of gene expression. Cell 159(4):925–939. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bandyra KJ, Said N, Pfeiffer V, Górna MW, Vogel J, Luisi BF (2012) The seed region of a small RNA drives the controlled destruction of the target mRNA by the Endoribonuclease RNase E. Mol Cell 47(6):943–953. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gorski SA, Vogel J, Doudna JA (2017) RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol 18(4):215–228. CrossRefPubMedGoogle Scholar
  22. 22.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. CrossRefPubMedGoogle Scholar
  23. 23.
    Zadeh JN, Steenberg CD, Bois JS, Wolfe BR, Pierce MB, Khan AR, Dirks RM, Pierce NA (2011) NUPACK: analysis and design of nucleic acid systems. J Comput Chem 32(1):170–173. CrossRefPubMedGoogle Scholar
  24. 24.
    Engler C, Marillonnet S (2014) Golden gate cloning. In: Valla S, Lale R (eds) DNA cloning and assembly methods, vol 1116. Humana Press, New York, pp 119–131CrossRefGoogle Scholar
  25. 25.
    Gottesman S, McCullen C, Guillier M, Vanderpool C, Majdalani N, Benhammou J, Thompson K, FitzGerald P, Sowa N, FitzGerald D (2006) Small RNA regulators and the bacterial response to stress. Cold Spring Harb Symp Quant Biol 71:1–11. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kostecki JS, Li H, Turner RJ, DeLisa MP (2010) Visualizing interactions along the Escherichia coli twin-arginine translocation pathway using protein fragment complementation. PLoS One 5(2):e9225. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Liu MY, Romeo T (1997) The global regulator CsrA of Escherichia coli is a specific mRNA-binding protein. J Bacteriol 179(14):4639–4642. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Edwards AN, Patterson-Fortin LM, Vakulskas CA, Mercante JW, Potrykus K, Vinella D, Camacho MI, Fields JA, Thompson SA, Georgellis D, Cashel M, Babitzke P, Romeo T (2011) Circuitry linking the Csr and stringent response global regulatory systems. Mol Microbiol 80(6):1561–1580. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Klein JS, Jiang S, Galimidi RP, Keeffe JR, Bjorkman PJ (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Eng Des Sel 27(10):325–330. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 60(3):512–538PubMedPubMedCentralGoogle Scholar
  31. 31.
    Xu L, Chen H, Hu X, Zhang R, Zhang Z, Luo ZW (2006) Average gene length is highly conserved in prokaryotes and eukaryotes and diverges only between the two kingdoms. Mol Biol Evol 23(6):1107–1108. CrossRefPubMedGoogle Scholar
  32. 32.
    Gama-Castro S, Salgado H, Santos-Zavaleta A, Ledezma-Tejeida D, Muñiz-Rascado L, García-Sotelo JS, Alquicira-Hernández K, Martínez-Flores I, Pannier L, Castro-Mondragón JA, Medina-Rivera A, Solano-Lira H, Bonavides-Martínez C, Pérez-Rueda E, Alquicira-Hernández S, Porrón-Sotelo L, López-Fuentes A, Hernández-Koutoucheva A, Moral-Chávez VD, Rinaldi F, Collado-Vides J (2016) RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond. Nucleic Acids Res 44(D1):D133–D143. CrossRefPubMedGoogle Scholar
  33. 33.
    Liu MY, Gui G, Wei B, Preston JF 3rd, Oakford L, Yuksel U, Giedroc DP, Romeo T (1997) The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli. J Biol Chem 272(28):17502–17510. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Abigail N. Leistra
    • 1
  • Mia K. Mihailovic
    • 1
  • Lydia M. Contreras
    • 1
    Email author
  1. 1.McKetta Department of Chemical EngineeringUniversity of Texas at AustinAustinUSA

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