Monitoring RNA-ligand interactions using isothermal titration calorimetry.
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.
Gilbert
Sunny D
SD
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Batey
Robert T
RT
D015075:2-Aminopurine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002151:Calorimetry / Q000379:methods*; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D017186:Titrimetry
D008024:Ligands; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D012313:RNA