Isothermal titration calorimetry of RNA

Abstract

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA.

Figure 1

A schematic diagram of a typical energy compensation isothermal titration calorimeter.

Figure 2

Example ITC data from an experiment measuring the energetics of RNA duplex formation. A 7-nucleotide single-stranded RNA was titrated into its complement in 50 mM HEPES pH 7.5, 1 M NaCl at 25 °C. Top Panel: Power versus time curve. Bottom Panel: Thermogram of the integrated peak intensities plotted against the molar ratio of the two strands. Fitting of this titration yielded a K_a = 4.3 × 10⁷ M⁻¹, ΔH = −46.1 ± 0.2 kcal/mol and n = 0.91 ± 0.01. Data reproduced with permission from ref.(24) (Copyright, American Chemical Society).

Figure 3

Overview of an ITC experiment.

Figure 4

Preparation of the ITC instrument for an experiment involving one or more RNA substrates.