Encyclopedia of Experiments: Biological Techniques
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Transcript
Differential scanning calorimetry helps study biomolecule-ligand interactions by measuring the heat required to denature the biomolecule in the absence and presence of the ligand.
To begin, take a reference containing free DNA aptamers — structured single-stranded oligonucleotides — and a sample containing thermolabile ligand-bound aptamers. The ligands stabilize the aptamer's secondary structure.
Perform a forward scan by heating the solutions. Upon reaching the melting temperature, the free aptamers denature.
The ligand-stabilized aptamers dissociate from the ligands and denature at a relatively higher temperature, causing an upshift in the melting temperature compared to the free aptamers. At high temperatures, a subset of ligands obtains an irreversible secondary form with a low binding affinity.
Next, perform a reverse scan by cooling the solutions in order to regain the aptamer structure. At the lower temperature, the initial and converted ligands in the sample bind to the aptamers.
During the second forward scan, the aptamers bound with a weak affinity to the converted ligands dissociate and denature at a lower temperature than the initial ligand-bound aptamers, causing a downshift in the melting temperature.
With successive scans, the ligands continue to convert to the secondary form, resulting in the complete depletion of the initial form. Obtain the thermogram.
With each scan, the ligand-bound aptamer's melting temperature shifts toward the free aptamer, indicating the weak binding affinity of the secondary form.
