Skip to main content
SpringerLink
Log in

Isothermal Titration Calorimetry: Assisted Crystallization of RNA–Ligand Complexes

  • Protocol
Book cover Nucleic Acid Crystallography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1320))

Abstract

The success rate of nucleic acids/ligands co-crystallization can be significantly improved by performing preliminary biophysical analyses. Among suitable biophysical approaches, isothermal titration calorimetry (ITC) is certainly a method of choice. ITC can be used in a wide range of experimental conditions to monitor in real time the formation of the RNA– or DNA–ligand complex, with the advantage of providing in addition the complete binding profile of the interaction. Following the ITC experiment, the complex is ready to be concentrated for crystallization trials. This chapter describes a detailed experimental protocol for using ITC as a tool for monitoring RNA/small molecule binding, followed by co-crystallization.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Feig AL (2007) Applications of isothermal titration calorimetry in RNA biochemistry and biophysics. Biopolymers 87:293–301

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Feig AL (2009) Studying RNA-RNA and RNA-protein interactions by isothermal titration calorimetry. Methods Enzymol 468:409–422

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Gilbert SD, Batey RT (2009) Monitoring RNA-ligand interactions using isothermal titration calorimetry. Methods Mol Biol 540:97–114

    Article  CAS  PubMed  Google Scholar 

  4. Salim NN, Feig AL (2009) Isothermal titration calorimetry of RNA. Methods 47:198–205

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Pilch DS, Kaul M, Barbieri CM, Kerrigan JE (2003) Thermodynamics of aminoglycoside-rRNA recognition. Biopolymers 70:58–79

    Article  CAS  PubMed  Google Scholar 

  6. Kulshina N, Edwards TE, Ferre-D'Amare AR (2010) Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch. RNA 16:186–196

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Pikovskaya O, Polonskaia A, Patel DJ, Serganov A (2011) Structural principles of nucleoside selectivity in a 2′-deoxyguanosine riboswitch. Nat Chem Biol 7:748–755

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Sokoloski JE, Dombrowski SE, Bevilacqua PC (2012) Thermodynamics of ligand binding to a heterogeneous RNA population in the malachite green aptamer. Biochemistry 51:565–572

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Ennifar E, Aslam MW, Strasser P, Hoffmann G, Dumas P, van Delft FL (2013) Structure-guided discovery of a novel aminoglycoside conjugate targeting HIV-1 RNA viral genome. ACS Chem Biol 8:2509–2517

    Article  CAS  PubMed  Google Scholar 

  10. Trausch JJ, Batey RT (2013) A disconnect between high-affinity binding and efficient regulation by antifolates and purines in the tetrahydrofolate riboswitch. Chem Biol 1(2):205–216

    Google Scholar 

  11. Datta K, LiCata VJ (2003) Thermodynamics of the binding of Thermus aquaticus DNA polymerase to primed-template DNA. Nucleic Acids Res 31:5590–5597

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Recht MI, Williamson JR (2004) RNA tertiary structure and cooperative assembly of a large ribonucleoprotein complex. J Mol Biol 344:395–407

    Article  CAS  PubMed  Google Scholar 

  13. Bauer WJ, Heath J, Jenkins JL, Kielkopf CL (2012) Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1. J Mol Biol 415:727–740

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Freisz S, Mezher J, Hafirassou L, Wolff P, Nomine Y, Romier C, Dumas P, Ennifar E (2012) Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein. RNA Biol 9:966–977

    Article  CAS  PubMed  Google Scholar 

  15. Bec G, Meyer B, Gerard MA, Steger J, Fauster K, Wolff P, Burnouf D, Micura R, Dumas P, Ennifar E (2013) Thermodynamics of HIV-1 reverse transcriptase in action elucidates the mechanism of action of non-nucleoside inhibitors. J Am Chem Soc 135:9743–9752

    Article  CAS  PubMed  Google Scholar 

  16. Lukavsky PJ, Daujotyte D, Tollervey JR, Ule J, Stuani C, Buratti E, Baralle FE, Damberger FF, Allain FH (2013) Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Nat Struct Mol Biol 20:1443–1449

    Article  CAS  PubMed  Google Scholar 

  17. Neuenfeldt A, Lorber B, Ennifar E, Gaudry A, Sauter C, Sissler M, Florentz C (2013) Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture. Nucleic Acids Res 41:2698–2708

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Vander Meulen KA, Davis JH, Foster TR, Record MT Jr, Butcher SE (2008) Thermodynamics and folding pathway of tetraloop receptor-mediated RNA helical packing. J Mol Biol 384:702–717

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Reymond C, Bisaillon M, Perreault JP (2009) Monitoring of an RNA multistep folding pathway by isothermal titration calorimetry. Biophys J 96:132–140

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Salim N, Lamichhane R, Zhao R, Banerjee T, Philip J, Rueda D, Feig AL (2012) Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex. Biophys J 102:1097–1107

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Zhang J, Ferre-D'Amare AR (2013) Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA. Nature 500:363–366

    Article  CAS  PubMed  Google Scholar 

  22. Ladbury JE, Chowdhry BZ (1996) Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem Biol 3:791–801

    Article  CAS  PubMed  Google Scholar 

  23. Leavitt S, Freire E (2001) Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol 11:560–566

    Article  CAS  PubMed  Google Scholar 

  24. Velazquez Campoy A, Freire E (2005) ITC in the post-genomic era…? Priceless. Biophys Chem 115:115–124

    Article  CAS  PubMed  Google Scholar 

  25. Privalov PL, Dragan AI (2007) Microcalorimetry of biological macromolecules. Biophys Chem 126:16–24

    Article  CAS  PubMed  Google Scholar 

  26. Ennifar E, Paillart JC, Marquet R, Ehresmann B, Ehresmann C, Dumas P, Walter P (2003) HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics. J Biol Chem 278:2723–2730

    Article  CAS  PubMed  Google Scholar 

  27. Ennifar E, Paillart JC, Bodlenner A, Walter P, Weibel JM, Aubertin AM, Pale P, Dumas P, Marquet R (2006) Targeting the dimerization initiation site of HIV-1 RNA with aminoglycosides: from crystal to cell. Nucleic Acids Res 34:2328–2339

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Bernacchi S, Freisz S, Maechling C, Spiess B, Marquet R, Dumas P, Ennifar E (2007) Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion. Nucleic Acids Res 35:7128–7139

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Ennifar E, Paillart JC, Bernacchi S, Walter P, Pale P, Decout JL, Marquet R, Dumas P (2007) A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site. Biochimie 89:1195–1203

    Article  CAS  PubMed  Google Scholar 

  30. Keller S, Vargas C, Zhao H, Piszczek G, Brautigam CA, Schuck P (2012) High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal Chem 84:5066–5073

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Bernacchi S, Ennifar E, Toth K, Walter P, Langowski J, Dumas P (2005) Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site. J Biol Chem 280:40112–40121

    Article  CAS  PubMed  Google Scholar 

  32. Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179:131–137

    Article  CAS  PubMed  Google Scholar 

  33. Tellinghuisen J (2005) Optimizing experimental parameters in isothermal titration calorimetry. J Phys Chem B 109:20027–20035

    Article  CAS  PubMed  Google Scholar 

  34. Tellinghuisen J (2008) Isothermal titration calorimetry at very low c. Anal Biochem 373:395–397

    Article  CAS  PubMed  Google Scholar 

  35. Burnouf D, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M, Dumas P (2012) kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J Am Chem Soc 134:559–565

    Article  CAS  PubMed  Google Scholar 

  36. Spolar RS, Record MT Jr (1994) Coupling of local folding to site-specific binding of proteins to DNA. Science 263:777–784

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Agence Nationale pour la Recherche (grant ANR-12-BS07-0007-03 “ClickEnARN”) and the Agence Nationale de Recherches sur le SIDA (ANRS). The authors would like to thank Vincent Olieric (Paul Scherrer Institute/Swiss Light Source, Villigen, Switzerland), Natalia Markova and Peter Gimeson (Microcal-Malvern, Uppsala, Sweden).

Author information

Authors and Affiliations

  1. Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire, UPR 9002 CNRS/Université de Strasbourg, 15 Rue René Descartes, Strasbourg, 67084, France

    Cyrielle Da Veiga, Joelle Mezher, Philippe Dumas & Eric Ennifar

Authors
  1. Cyrielle Da Veiga
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joelle Mezher
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Philippe Dumas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eric Ennifar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eric Ennifar .

Editor information

Editors and Affiliations

  1. CNRS Inst de Biol Moléculaire et Cellulaire, Strasbourg, France

    Eric Ennifar

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Da Veiga, C., Mezher, J., Dumas, P., Ennifar, E. (2016). Isothermal Titration Calorimetry: Assisted Crystallization of RNA–Ligand Complexes. In: Ennifar, E. (eds) Nucleic Acid Crystallography. Methods in Molecular Biology, vol 1320. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2763-0_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2763-0_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2762-3

  • Online ISBN: 978-1-4939-2763-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation