Systems Biophysics, Department of Physics, Ludwig-Maximilians-Universität München and Center for NanoScience, Amalienstasse 54, 80799, München, Germany.
Angew Chem Int Ed Engl. 2021 Jun 14;60(25):13988-13995. doi: 10.1002/anie.202101261. Epub 2021 May 11.
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 -10 m s and 10 -10 s , 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.
微尺度热泳（MST）是一种基于分子在温度梯度中定向运动来测量结合物-配体系统结合亲和力的多功能技术。我们通过增加样品的热耗散，将 MST 扩展为在单个实验中测量结合动力学和结合亲和力。动力学弛豫指纹是从局部加热后样品热力学再平衡过程中的荧光变化中得出的。使用这种方法，我们测量了在 10 -10 m s 和 10 -10 s 范围内的 DNA 杂交的上结合速率和下结合速率。我们观察到 DNA 杂交解速率与盐浓度、链长和倒数温度的预期指数依赖性。测量得到的上结合速率与盐浓度呈线性关系，与链长和温度的依赖性较弱。对于具有大焓贡献的生物分子相互作用，即使在拥挤的溶液中，动力学 MST 技术也提供了一种稳健、经济高效且无需固定化的方法来同时确定动力学速率和结合亲和力。
