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Riboswitches pp 97–114Cite as

Monitoring RNA–Ligand Interactions Using Isothermal Titration Calorimetry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 540))

Summary

Isothermal titration calorimetry (ITC) is a biophysical technique that measures the heat evolved or absorbed during a reaction to report the enthalpy, entropy, stoichiometry of binding, and equilibrium association constant. A significant advantage of ITC over other methods is that it can be readily applied to almost any RNA–ligand complex without having to label either molecule and can be performed under a broad range of pH, temperature, and ionic concentrations. During our application of ITC to investigate the thermodynamic details of the interaction of a variety of compounds with the purine riboswitch, we have explored and optimized experimental parameters that yield the most useful and reproducible results for RNAs. In this chapter, we detail this method using the titration of an adenine-binding RNA with 2,6-diaminopurine (DAP) as a practical example. Our insights should be generally applicable to observing the interactions of a broad range of molecules with structured RNAs.

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Acknowledgments

The authors would like to thank Deborah Wuttke and Jonas Fast for useful discussions on optimizing ITC experiments. This work was made possible by a Research Scholar Grant from the American Cancer Society to R.T.B.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 80309-0215, USA

    Sunny D. Gilbert & Robert T. Batey

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  1. Sunny D. Gilbert
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  2. Robert T. Batey
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Correspondence to Robert T. Batey .

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    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

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    Gilbert, S., Batey, R. (2009). Monitoring RNA–Ligand Interactions Using Isothermal Titration Calorimetry . In: Serganov, A. (eds) Riboswitches. Methods in Molecular Biology, vol 540. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-558-9_8

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    • DOI: https://doi.org/10.1007/978-1-59745-558-9_8

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    • Publisher Name: Humana Press, Totowa, NJ

    • Print ISBN: 978-1-934115-88-6

    • Online ISBN: 978-1-59745-558-9

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