Stein, Julian A. C.; Ianeselli, Alan; Braun, Dieter  ORCID: 0000-0001-7751-1448
(2021):
Kinetic microscale thermophoresis.
In: Angewandte Chemie
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Abstract
We established an extension of Microscale Thermophoresis (MST) to measure binding kinetics together with binding affinity in a single experimental run, by increasing the thermal dissipation of the sample. After the switch-off of an IR laser, that locally heated the sample, the temperature re-equilibrated within 250 ms. The kinetic relaxation fingerprints were extracted from the fluorescence changes back to thermodynamic equilibrium. We measured DNA hybridization on-rates and off-rates in the range between 104-106 M-1s-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 and weak if no dependence at all on length and temperature. For biological binding reactions with sufficient enthalpic contributions, Kinetic MST offers a robust and immobilization-free determination of kinetic rates and binding affinity and also in crowded solutions.
