Journal
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
Volume 60, Issue 25, Pages 13988-13995Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anie.202101261
Keywords
binding kinetics; DNA hybridization; DNA thermodynamics; kinetic rates; microscale thermophoresis
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Funding
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [364653263-TRR 235 P11]
- Simons Collaboration on the Origins of Life [327125FY19]
- Projekt DEAL
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MST is a versatile technique for measuring binding affinities, based on directional movement of molecules in a temperature gradient. We extended MST to measure binding kinetics by increasing thermal dissipation, allowing for simultaneous determination of kinetics and binding affinity. By measuring DNA hybridization rates, we observed linear dependence of on-rates on salt concentration and weak dependence on strand length and temperature.
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.
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