Abstract
Fluorescence anisotropy provides a powerful method to quantitatively characterize kinetic and thermodynamic parameters for many RNA–ligand interactions. By manipulating assay design, it is possible to extract equilibrium binding constants down to the low or even sub-nanomolar range, as well as measure reaction on- and off-rates, changes in enthalpy and entropy, and RNA-binding site size. These tools are invaluable for comparing and contrasting potential mechanisms of RNA recognition by proteins or other binding partners. In this chapter, we review the theory behind the technique and describe some recent applications. We explain how to generate binding isotherms using fluorescence anisotropy, including discussion of the instrumentation and fluorescent substrates required. To analyze isotherms, we derive relationships between anisotropy and free protein concentration for a variety of reaction mechanisms and discuss statistical validation methods. Finally, we have included sections on the use of plate readers for measuring fluorescence anisotropy across large numbers of samples and their application to high-throughput drug screening. Fluorescence anisotropy has been used to elucidate mechanisms involved in many critical processes in RNA biology, including assembly of the translation initiation complex and recognition of mRNAs targeted for degradation.
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References
Albinsson B, Ericksson S, Lyng R, Kubista M (1991) The electronically excited states of 2-phenylindole. Chem Phys 151:157
Anderson BJ, Larkin C, Guja K, Schildbach JF (2008) Using fluorophore-labeled oligonucleotides to measure affinities of protein-DNA interactions. Methods Enzymol 450:253–272
Baumann C, Otridge J, Gollnick P (1996) Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA. J Biol Chem 271:12269–12274
Beaudette NV, Langerman N (1980) The thermodynamics of nucleotide binding to proteins. CRC Crit Rev Biochem 9:145–169
Beechem JM, Brand L (1985) Time-resolved fluorescence of proteins. Annu Rev Biochem 54:43–71
Bernstein J, Patterson DN, Wilson GM, Toth EA (2008) Characterization of the essential activities of Saccharomyces cerevisiae Mtr4p, a 3′ to 5′ helicase partner of the nuclear exosome. J Biol Chem 283:4930–4942
Bujalowski W, Jezewska MJ (2000) Quantitative determination of equilibrium binding isotherms for multiple ligand-macromolecule interactions using spectroscopic methods. In: Gore MG (ed) Spectrophotometry and spectrofluorimetry. Oxford University Press, Oxford, UK, pp 141–165
Draper DE (1995) Protein-RNA recognition. Annu Rev Biochem 64:593–620
Eversole A, Maizels N (2000) In vitro properties of the conserved mammalian protein hnRNPD suggest a role in telomere maintenance. Mol Cell Biol 20:5425–5432
Fernandes PB (1998) Technological advances in high-throughput screening. Curr Opin Chem Biol 2:597–603
Fialcowitz-White EJ, Brewer BY, Ballin JD, Willis CD, Toth EA, Wilson GM (2007) Specific protein domains mediate cooperative assembly of HuR oligomers on AU-rich mRNA-destabilizing sequences. J Biol Chem 282:20948–20959
Gradinaru CC, Marushchak DO, Samim M, Krull UJ (2010) Fluorescence anisotropy: from single molecules to live cells. Analyst 135:452–459
Gupta YK, Lee TH, Edwards TA, Escalante CR, Kadyrova LY, Wharton RP, Aggarwal AK (2009) Co-occupancy of two Pumilio molecules on a single hunchback NRE. RNA 15:1029–1035
Ha JH, Spolar RS, Record MT Jr (1989) Role of the hydrophobic effect in stability of site-specific protein-DNA complexes. J Mol Biol 209:801–816
Huang X (2003) Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand. J Biomol Screen 8:34–38
Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110:2685–2708
Jameson DM, Sawyer WH (1995) Fluorescence anisotropy applied to biomolecular interactions. Methods Enzymol 246:283–300
Lakowicz JR (1999) Fluorescence anisotropy. In: Principles of fluorescence spectroscopy. Kluwer, New York, pp 291–319
Liao B, Hu Y, Brewer G (2007) Competitive binding of AUF1 and TIAR to MYC mRNA controls its translation. Nat Struct Mol Biol 14:511–518
Lohman TM, Bujalowski W (1991) Thermodynamic methods for model-independent determination of equilibrium binding isotherms for protein-DNA interactions: spectroscopic approaches to monitor binding. Methods Enzymol 208:258–290
Lu JY, Sadri N, Schneider RJ (2006) Endotoxic shock in AUF1 knockout mice mediated by failure to degrade proinflammatory cytokine mRNAs. Genes Dev 20:3174–3184
Maag D, Lorsch JR (2003) Communication between eukaryotic translation initiation factors 1 and 1A on the yeast small ribosomal subunit. J Mol Biol 330:917–924
Mao C, Flavin KG, Wang S, Dodson R, Ross J, Shapiro DJ (2006) Analysis of RNA-protein interactions by a microplate-based fluorescence anisotropy assay. Anal Biochem 350:222–232
Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK (1997) Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89:951–961
Meisner NC, Hintersteiner M, Mueller K, Bauer R, Seifert JM, Naegeli HU, Ottl J, Oberer L, Guenat C, Moss S, Harrer N, Woisetschlaeger M, Buehler C, Uhl V, Auer M (2007) Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR. Nat Chem Biol 3:508–515
Mitchell SF, Walker SE, Algire MA, Park EH, Hinnebusch AG, Lorsch JR (2010) The 5′-7-methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol Cell 39:950–962
Owicki JC (2000) Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J Biomol Screen 5:297–306
Ranji A, Shkriabai N, Kvaratskhelia M, Musier-Forsyth K, Boris-Lawrie K (2011) Features of double-stranded RNA-binding domains of RNA helicase A are necessary for selective recognitions and translation of complex mRNAs. J Biol Chem 286:5328–5337
Sadana A (2001) A kinetic study of analyte-receptor binding and dissociation, and dissociation alone, for biosensor applications: a fractal analysis. Anal Biochem 291:34–47
Sarkar S, Han J, Sinsimer KS, Liao B, Foster RL, Brewer G, Pestka S (2011) RNA-binding protein AUF1 regulates lipopolysaccharide-induced IL-10 expression by activating IκB kinase complex in monocytes. Mol Cell Biol 31:602–615
Wagner BJ, DeMaria CT, Sun Y, Wilson GM, Brewer G (1998) Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms. Genomics 48:195–202
Wilson GM (2005) RNA folding and RNA-protein binding analyzed by fluorescence anisotropy and resonance energy transfer. In: Geddes CD, Lakowicz JR (eds) Reviews in fluorescence, vol 2. Springer, New York, pp 223–243
Wilson GM, Sutphen K, Bolikal S, Chuang K, Brewer G (2001) Thermodynamics and kinetics of Hsp70 association with A + U-rich mRNA-destabilizing sequences. J Biol Chem 276:44450–44456
Wilson GM, Lu J, Sutphen K, Suarez Y, Sinha S, Brewer B, Villanueva-Feliciano EC, Ylsa RM, Charles S, Brewer G (2003) Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure. J Biol Chem 278:33039–33048
Yan Y, Marriott G (2003) Analysis of protein interactions using fluorescence technologies. Curr Opin Chem Biol 7:635–640
Yumak H, Khan MA, Goss DJ (2010) Poly(A) tail affects equilibrium and thermodynamic behavior of tobacco etch virus mRNA with translation initiation factors elF4F, elF4B and PABP. Biochim Biophys Acta 1799:653–658
Zhang JH, Chung T, Oldenburg K (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73
Zucconi BE, Ballin JD, Brewer BY, Ross CR, Huang J, Toth EA, Wilson GM (2010) Alternatively expressed domains of AU-rich element RNA-binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands. J Biol Chem 285:39127–39139
Acknowledgements
This work was supported by NIH grants CA052443 (to G.B.) and CA102428 (to G.M.W.).
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Kishor, A., Brewer, G., Wilson, G.M. (2012). Analyses of RNA–Ligand Interactions by Fluorescence Anisotropy. In: Dinman, J. (eds) Biophysical approaches to translational control of gene expression. Biophysics for the Life Sciences, vol 1. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3991-2_9
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DOI: https://doi.org/10.1007/978-1-4614-3991-2_9
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