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Kinetic Microscale Thermophoresis for Simultaneous Measurement of Binding Affinity and Kinetics
Author(s) -
Stein Julian A. C.,
Ianeselli Alan,
Braun Dieter
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202101261
Subject(s) - microscale thermophoresis , kinetics , microscale chemistry , chemistry , thermophoresis , kinetic energy , receptor–ligand kinetics , thermodynamics , relaxation (psychology) , diffusion , analytical chemistry (journal) , thermal , chromatography , psychology , biochemistry , physics , mathematics education , mathematics , quantum mechanics , nanofluid , social psychology
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 10 4 –10 6 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.