Isothermal titration calorimetry

ITC binding experiments were performed using a MicroCal VP-ITC instrument in a manner similar to what we previously described³⁵. Samples were degassed before analysis with a MicroCal Thermo Vac unit. All experiments were corrected for the heat of dilution of the titrant. Titrations were performed with the aptamer samples in the cell and the ligand as the titrant, in the needle. All aptamer samples were heated in a 95 °C water bath for 3 min and cooled in an ice water bath prior to use in a binding experiment to allow the DNA aptamer to anneal in an intramolecular fashion.

The binding experiments were performed at 20 °C with the aptamer solution at a concentration of 10 to 100 µM using adenosine concentrations of 0.312 to 2.8 mM. Apt1d, Apt1d-GC1 and Apt1d-GC2 samples were run at 100 µM DNA at 20 °C. All binding experiments consisted of an initial delay of 60 s, a first injection of 2 µL and then a 300 s delay. Subsequent 34 injections were 8 µL, spaced every 300 s. The first point was removed from all data sets due to the different injection volume and delay parameters.

ITC data was fit to both cooperative and two-independent sites binding models described by Freiburger et al.⁵ using MATLAB 14 software. Data following a one-set of sites model was analyzed using the manufacturer provided Origin 7.0 software. The ITC data at aptamer concentrations of 70 µM for ATP3, ATP6, ATP17 and ATP7 were analysed to determine the Hill coefficient (n_H) using established methods^5,36,37.