Abstract
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.
Highlights
The dissociation constant Kd = koff/kon characterizes the binding affinity of a binder–ligand system and has been extensively studied in many research fields.[1,2,3,4] Kd is usually determined by the analysis of equilibrated states of binder– ligand systems
We extended the conventional Microscale thermophoresis (MST) setup (Nanotemper Monolith NT.115Pico) to kinetic MST by placing the samplecontaining capillary on a silicon wafer and immersing it in oil (Figure 1)
We have shown that combining MST with the temperature jump technique provides a novel method to determine the kinetic rates along with binding affinities in a single experiment
Summary
KMST is an extension of the well-established and widely used microscale thermophoresis (MST) method.[1,21,22,23,24,25,26,27] MST uses binding-dependent intensity change of fluorescently labeled molecules in a microscopic temperature gradient to measure the binding affinity. Analysis of the temperature-dependent features, including the bleaching, diffusion, thermophoretic and kinetic contribution to the fluorescence intensity (Figure 1 and Figure 2), allows for the determination of the binding affinity and the kinetic rates in a single experiment (Figure 3). Our results on DNA hybridization show that KMST is a promising method to measure reaction kinetics without immobilization, with fluorescent labeling of only one binding partner and in crowded solutions (Figure 6)
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