Fluorescence Anisotropy-Based Salt-Titration Approach to Characterize Protein–Nucleic Acid Interactions

  • Tiffiny Rye-McCurdy
  • Ioulia Rouzina
  • Karin Musier-ForsythEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)


Many proteins bind nucleic acids (NA) via cationic residues that interact electrostatically with the anionic phosphate backbone of RNA or DNA. These electrostatic interactions are often insensitive to NA sequence and structure, but confer strong salt dependence to the binding interactions. In contrast, salt-independent non-electrostatic contacts reflect more specific binding interactions. Proteins with multiple cationic NA-binding domains connected by flexible linkers, such as the HIV-1 Gag polyprotein, may bind different NA molecules in distinct ways. For example, Gag binding to the Psi-packaging signal of the HIV-1 RNA genome optimizes the specific non-electrostatic binding component of this protein–RNA interaction. In contrast, Gag binding to a non-psi RNA optimizes the electrostatic interactions at the expense of specific contacts. Here, we describe a fluorescence anisotropy-based salt-titration approach that allows complete characterization of both electrostatic and non-electrostatic binding components for any protein–NA complex in a quantitative manner within a single assay.

Key words

Fluorescence anisotropy Proteins Nucleic acids Specific binding Nonspecific binding Electrostatic interactions Non-electrostatic interactions HIV-1 Gag Nucleocapsid 



We thank Mr. Joseph A. Webb and Drs. Christopher Jones and Leslie Parent for their role in FA-based salt-titration method development and application, Ms. Roopa Comandur for determining the effect of glycerol on the FA signal, and Mr. Erik Olson and Ms. Weixin Wu for critical reading of this chapter prior to submission. This work was supported by NIH GM065056 (K.M.-F.) and T.R.-M. was supported by NIH T32-GM008512.


  1. 1.
    Luger K, Mader AW, Richmond RK et al (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251–260PubMedCrossRefGoogle Scholar
  2. 2.
    Gansen A, Valeri A, Hauger F et al (2009) Nucleosome disassembly intermediates characterized by single-molecule FRET. Proc Natl Acad Sci U S A 106:15308–15313PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Andrews AJ, Luger K (2011) A coupled equilibrium approach to study nucleosome thermodynamics. Methods Enzymol 488:265–285PubMedCrossRefGoogle Scholar
  4. 4.
    Moyle-Heyrman G, Zaichuk T, Xi L et al (2013) Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning. Proc Natl Acad Sci U S A 110:20158–20163PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Sidorova NY, Muradymov S, Rau DC (2011) Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV. FEBS J 278:2713–2727PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Rau DC, Sidorova NY (2010) Diffusion of the restriction nuclease EcoRI along DNA. J Mol Biol 395:408–416PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Datta SA, Rein A (2009) Preparation of recombinant HIV-1 gag protein and assembly of virus-like particles in vitro. Methods Mol Biol 485:197–208PubMedCrossRefGoogle Scholar
  8. 8.
    Rein A, Datta SA, Jones CP et al (2011) Diverse interactions of retroviral Gag proteins with RNAs. Trends Biochem Sci 36:373–380PubMedCentralPubMedGoogle Scholar
  9. 9.
    O’Carroll IP, Soheilian F, Kamata A et al (2013) Elements in HIV-1 Gag contributing to virus particle assembly. Virus Res 171:341–345PubMedCrossRefGoogle Scholar
  10. 10.
    Datta SA, Curtis JE, Ratcliff W et al (2007) Conformation of the HIV-1 Gag protein in solution. J Mol Biol 365:812–824PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Munro JB, Nath A, Farber M et al (2014) A conformational transition observed in single HIV-1 Gag molecules during in vitro assembly of virus-like particles. J Virol 88:3577–3585PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Coffin JM, Hughes SH, Varmus H (1997) Retroviruses. Cold Spring Harbor Laboratory Press, Plainview, xv, 843 pGoogle Scholar
  13. 13.
    Darlix JL, Godet J, Ivanyi-Nagy R et al (2011) Flexible nature and specific functions of the HIV-1 nucleocapsid protein. J Mol Biol 410:565–581PubMedCrossRefGoogle Scholar
  14. 14.
    Levin JG, Mitra M, Mascarenhas A et al (2010) Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription. RNA Biol 7:754–774PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Sun M, Grigsby IF, Gorelick RJ et al (2014) Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging. J Virol 88:1271–1280PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Jones CP, Datta SA, Rein A et al (2011) Matrix domain modulates HIV-1 Gag’s nucleic acid chaperone activity via inositol phosphate binding. J Virol 85:1594–1603PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Chukkapalli V, Oh SJ, Ono A (2010) Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain. Proc Natl Acad Sci U S A 107:1600–1605PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Inlora J, Chukkapalli V, Derse D et al (2011) Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag. J Virol 85:3802–3810PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Alfadhli A, McNett H, Tsagli S et al (2011) HIV-1 matrix protein binding to RNA. J Mol Biol 410:653–666PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Vuilleumier C, Bombarda E, Morellet N et al (1999) Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study. Biochemistry 38:16816–16825PubMedCrossRefGoogle Scholar
  21. 21.
    Athavale SS, Ouyang W, McPike MP et al (2010) Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA. Biochemistry 49:3525–3533PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Record MTJ, Lohman TM, de Haseth PL (1976) Ion effects on ligand-nucleic acid interactions. J Mol Biol 107:145–158PubMedCrossRefGoogle Scholar
  23. 23.
    Record MTJ, Zhang W, Anderson CF (1998) Analysis of effects of salts and uncharged solutes on protein and nucleic acid equilibria and processes: a practical guide to recognizing and interpreting polyelectrolyte effects, Hofmeister effects, and osmotic effects of salts. Adv Protein Chem 51:281–353PubMedCrossRefGoogle Scholar
  24. 24.
    Manning GS (1978) The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q Rev Biophys 11:179–246PubMedCrossRefGoogle Scholar
  25. 25.
    Rouzina I, Bloomfield VA (1996) Influence of ligand spatial organization on competitive electrostatic binding to DNA. J Phys Chem 100:4305–4313CrossRefGoogle Scholar
  26. 26.
    Rouzina I, Bloomfield VA (1996) Competitive electrostatic binding of charged ligands to polyelectrolytes: planar and cylindrical geometries. J Phys Chem 100:4292–4304CrossRefGoogle Scholar
  27. 27.
    Rouzina I, Bloomfield VA (1997) Competitive electrostatic binding of charged ligands to polyelectrolytes: practical approach using the non-linear Poisson-Boltzmann equation. Biophys Chem 64:139–155PubMedCrossRefGoogle Scholar
  28. 28.
    Webb JA, Jones CP, Parent LJ et al (2013) Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging. RNA 19:1078–1088PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Mély Y, de Rocquigny H, Sorinas-Jimeno M et al (1995) Binding of the HIV-1 nucleocapsid protein to the primer tRNA(3Lys), in vitro, is essentially not specific. J Biol Chem 270:1650–1656PubMedCrossRefGoogle Scholar
  30. 30.
    Urbaneja MA, Kane BP, Johnson DG et al (1999) Binding properties of the human immunodeficiency virus type 1 nucleocapsid protein p7 to a model RNA: elucidation of the structural determinants for function. J Mol Biol 287:59–75PubMedCrossRefGoogle Scholar
  31. 31.
    Vo MN, Barany G, Rouzina I et al (2009) Effect of Mg(2+) and Na(+) on the nucleic acid chaperone activity of HIV-1 nucleocapsid protein: implications for reverse transcription. J Mol Biol 386:773–788PubMedCrossRefGoogle Scholar
  32. 32.
    Pagano JM, Farley BM, McCoig LM et al (2007) Molecular basis of RNA recognition by the embryonic polarity determinant MEX-5. J Biol Chem 282:8883–8894PubMedCrossRefGoogle Scholar
  33. 33.
    Chaudhuri AR, de Waal EM, Pierce A et al (2006) Detection of protein carbonyls in aging liver tissue: a fluorescence-based proteomic approach. Mech Ageing Dev 127:849–861PubMedCrossRefGoogle Scholar
  34. 34.
    Jones CP, Cantara WA, Olson ED et al (2014) Small-angle X-ray scattering-derived structure of the HIV-1 5′ UTR reveals 3D tRNA mimicry. Proc Natl Acad Sci U S A 111:3395–3400PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Norman DG, Grainger RJ, Uhrin D et al (2000) Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies. Biochemistry 39:6317–6324PubMedCrossRefGoogle Scholar
  36. 36.
    Ryder SP, Recht MI, Williamson JR (2008) Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol 488:99–115PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Pope AJ, Haupts UM, Moore KJ (1999) Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. Drug Discov Today 4:350–362PubMedCrossRefGoogle Scholar
  38. 38.
    Jing M, Bowser MT (2011) Methods for measuring aptamer-protein equilibria: a review. Anal Chim Acta 686:9–18PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New YorkCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tiffiny Rye-McCurdy
    • 1
  • Ioulia Rouzina
    • 2
  • Karin Musier-Forsyth
    • 1
    Email author
  1. 1.Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Centers for Retroviral Research and RNA BiologyThe Ohio State UniversityColumbusUSA
  2. 2.Department of Biochemistry, Molecular Biology and BiophysicsUniversity of MinnesotaMinneapolisUSA

Personalised recommendations