Aptamer binding assays:
Surface Plasmon Resonance imaging (SPRi)
For large molecules we perform binding assays with surface plasmon resonance imaging (SPRi) system on a Horiba OpenPlex instrument.
This instrument differs from BiaCore analysis. We can analyze binding to multiple spots separately and simultaneously. Thiolated aptamers are spotted onto a gold chip on a glass prism. The target molecule flows over the immobilized aptamers and determine the rate of complex formation (ka) and the rate of complex disassociation (kd). We can determine binding affinity of antisense molecules and measure competition between aptamers for binding sites on proteins.
Isothermal Titration Calorimetry (ITC)
For small molecules we perform binding assays by Isothermal titration calorimetry (ITC) analysis on a Nano ITC by TA instruments.
The instrument contains two chambers, a reference chamber and a loading chamber.
The amount of energy required to maintain the temperature of the loading chamber at the same level as the reference chamber is measured in uJ/sec. The loading chamber is loaded with one of the components of the binding reaction under analysis (usually the aptamer). Then another component (usually the target) is serially injected into the loading chamber and the heat dynamics of the reaction are measured. The loading chamber has a gold surface thus we cannot use any target molecules that bind significantly to gold. We use relatively high concentrations of targets (low mM) in order to obtain reasonable heat of binding signals. There is also a need to run several negative controls.