Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction

Degas the buffer, biomolecule, and ligand solutions in a table top degasser prior to loading into the differential scanning calorimeter, or DSC. Unscrew the pressure handle from the DSC. Then, run silicone tubing from the working buffer, and attach it to the front flange of the reference capillary.

Create a bridge between the reference and sample capillaries by connecting the rear reference flange to the front sample flange. Next, attach a piece of silicone tubing to the rear sample flange that runs to a waste flask with a vacuum line attached. Turn on the vacuum line to flush the DSC with 200 milliliters of working buffer.

Begin by attaching roughly 3- to 5-centimeter sections of silicone tubing to the reference capillary flanges. Next, insert a 1-milliliter pipette tip into the silicone tubing of the rear flange. Draw 0.8 milliliters of working buffer with a pipette, and insert the pipette tip with buffer into the silicone tubing of the front reference flange.

Gently press the pipette plunger down to pass the working buffer through the front silicone tubing into the reference capillary and up into the attached pipette tip of the rear flange. Press the pipette plunger down until the working buffer level reaches just above the front silicone tubing. Then, release the pipette plunger until the working buffer level reaches just above the rear silicone tubing. Continue passing the working buffer back and forth in the reference capillary to purge the volume of bubbles.

Next, cap the rear pipette tip with a forefinger and gently pull up on the rear pipette tip and front pipette to remove them from the reference flanges with the silicone tubing attached. Load the sample capillary with working buffer as before.

Place a black plastic cap on the rear reference and sample flanges, leaving the front flanges uncovered. Attach the pressure handle to the DSC and then, open the DSC software and pressurize the instrument by clicking the red "Up" arrow at the top of the interface once the power reading has stabilized. The DSC power is indicated in a box at the top right of the interface, along with the instrument temperature and pressure reading.

Equilibrate the DSC with working buffer by performing a forward and reverse scan. In the "Experimental Method" tab on the left side of the screen, ensure that the "Scanning" option is selected to run the DSC in temperature-scanning mode.

In the "Temperature parameters" inset under the "Experimental Method" tab, click the button for heating. Enter 1 and 100 degrees Celsius for the lower and upper experimental temperatures, 1 degree Celsius per minute for the scan rate, and 60 seconds for the equilibration period. Click the "Add Series" button under the input field for the equilibration period. Enter 2 into the "Steps to add" field in the pop-up window and check the "Alternate Heating/Cooling" box. Click "OK."

The added scans appear in the lower portion of the interface. Check that the parameters for each scan are as desired. Start the experiment by clicking the green "Play" button at the top of the interface. Navigate to the desired window and input a file name for saving the experiment in the pop-up window. View the experiment progress by clicking the "Data" tab to the right of the "Experiment Method" tab.

Run reference experiments for baseline subtraction of the sample data by reloading the DSC with working buffer in both capillaries. Collect multiple forward and reverse scans over a suitable temperature range at 1 degree Celsius per minute with an upper equilibration time of 120 seconds.

Delete the previous buffer equilibration scans from the lower portion of the interface by highlighting each individually and clicking the red "X" to the middle right of the interface.

Add the new scans by clicking the "Add Series" button, entering 20 in the field for "Steps to add" and checking the "Alternate Heating/Cooling" box. Then, click "OK" and run the experiment by clicking the green "Play" button as before.

Repeat these steps with working buffer containing the desired concentration of ligand in both capillaries to obtain the reference experiments for the ligand. Collect two separate experiments to be used in acquiring the rate constant for thermolabile ligand conversion.

For the free biomolecule data set, ensured that the reference capillary contains the working buffer, while the sample capillary contains the free biomolecule at the desired concentration in working buffer. Next, run the sample experiments using the same DSC-loading procedure and experimental parameters used in the reference scans.

For the ligand-bound experiments, ensure that the ligand is in the working buffer in the reference capillary and the biomolecule plus ligand are in the working buffer in the sample capillary. Flush the system between additions of different ligands as before.

Perform one additional experiment with the biomolecule bound to the thermolabile ligand where the high-temperature equilibration period is increased to 600 seconds, and all other experimental parameters are the same as before. Proceed to data processing and analysis as described in the text protocol.