Microscale thermophoresis (MST) is a versatile technique to measure binding affinities of binder-ligand systems, based on the directional movement of molecules in a temperature gradient. We extended MST to measure binding kinetics as well as binding affinity in a single experiment by increasing the thermal dissipation of the sample. The kinetic relaxation fingerprints were derived from the fluorescence changes during thermodynamic re-equilibration of the sample after local heating. Using this method, we measured DNA hybridization on-rates and off-rates in the range 104 -106 m-1 s-1 and 10-4 -10-1 s-1 , respectively. We observed the expected exponential dependence of the DNA hybridization off-rates on salt concentration, strand length and inverse temperature. The measured on-rates showed a linear dependence on salt concentration and weak dependence on strand length and temperature. For biomolecular interactions with large enthalpic contributions, the kinetic MST technique offers a robust, cost-effective and immobilization-free determination of kinetic rates and binding affinity simultaneously, even in crowded solutions.
Keywords: DNA hybridization; DNA thermodynamics; binding kinetics; kinetic rates; microscale thermophoresis.
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