This protocol allows researchers to determine the binding affinities of aptamers against targets free in solution, while providing insight into the mechanism of binding. Because ITC measures changes in heat due to binding, neither aptamer nor ligand needs to be labeled or functionalized, as would be the case for techniques such as SPR. New users should pay close attention to matching buffers across all binding components.
Additionally, be aware that the technique has high sample requirements that may be difficult to meet for larger ligands such as proteins. To begin, activate the membrane of the dialysis column, fill with PBS buffer, equilibrate for 10 minutes at room temperature, and centrifuge at 5, 000 times G for 15 minutes. Remove the buffer and load 500 microliters of aptamer samples into the column.
Centrifuge at 5, 000 times G and repeat it four times to exchange the original buffer for PBS. Collect the dialyzed DNA aptamer using a pipette and transfer it to the new 1.5-milliliter tube. Collect the last flow-through buffer to dissolve tetracycline.
Ensure that the buffer for the experiment in the syringe matches the buffer in the reference cell. Determine the aptamer concentration again using a UV-visible spectrometer. Use the last exchange buffer to adjust the concentration to 40-micromolar tetracycline and two-micromolar aptamer.
Fold the DNA aptamer by heating at 90 degrees Celsius for 10 minutes, cooling at four degrees Celsius for 10 minutes, and then returning to room temperature for 20 minutes. Degas the folded aptamer and dialyzed tetracycline using a degassing station or vacuum pump for 25 minutes to eliminate dissolved gases. After cleaning up and confirming the cleanliness of the machine, test the accuracy of the machine with a standard kit that includes EDTA and calcium chloride using the default program and following the instructions from the manufacturer.
Set up the running parameters. Check the required volumes using a running program calculator. With this running parameter, perform ITC measurement with 230 microliters of 40-micromolar tetracycline in the ITC syringe and 485 microliters of two-micromolar aptamer in the ITC sample cell using the ITC.
Load the dialyzed tetracycline syringe plates and the folded aptamer into the sample cell, avoiding bubbles, using a pipette. Start running the ITC instrument by clicking on the Start button on the software. Open the data analysis software by double clicking to start analyzing the data.
Open the path of the saved raw data to know the tendency of binding. Open the Modeling tab and use different binding models to find the best fit for the data curve. Then, the software automatically calculates the ITC thermogram and various thermodynamic parameters, including enthalpy, entropy, free energy, equilibrium binding constant, and stoichiometry.
Collect the thermodynamic parameters determined from the data and fitting model information. Create a report including pictures of the ITC thermogram and various thermodynamic parameters. The aptamer selected by Kim, et al binds to tetracycline with binding affinities of disassociation constant one equals 13 micromolar and disassociation constant two equals 53 nanomolar.
The fitting model and stoichiometry from ITC reflect that the aptamer binds to tetracycline through a 2:1 binding ratio with the sequential binding model. The thermodynamic parameters determined by ITC measurement for site two indicated that enthalpy overcoming relatively significant entropic loss drives the strong binding. The enthalpy-driven binding with entropy loss relates to nucleic acid conformational changes, which have been reported as binding behaviors between RNA and a small molecule.
The aptamer and ligand must be in the same buffer. The aptamer should be refolded if developed with refolding, and samples must be degassed. Addressing these considerations will ensure that only aptamer-ligand interactions contribute to the measured signal.
Due to the size of the aptamer target, an appropriate follow-up method could be microscale thermophoresis to confirm the measured binding affinity free in solution.
