New method detects binding interactions in blood samples
Chemical compounds with low molecular weights ("small molecules") are ideal starting points for the development of novel pharmacological agents. Their small size facilitates entry into cells, where they must act, usually by binding to proteins that are much larger than they are. A research team consisting of LMU biophysicist Professor Dieter Braun and scientists at NanoTemper Technologies GmbH, an LMU spin-off, have developed a new method that can detect and quantify interactions between molecules of widely different sizes. The technique makes use of “Microscale Thermophoresis” (MST), which is sensitive to the electrical charge of molecule changes of the hydration spheres (bound layers of water) associated with them. “We can now analyze binding interactions between molecules, essentially irrespective of the size differences between the partners involved”, says Braun. “This allows us to monitor the binding of ions or small-molecule interactors to proteins, and by using fluorescent dyes as markers, we can study binding in blood and other complex biological fluids.” The new method was developed in cooperation with NanoTemper Technologies, and the study was supported by the Cluster of Excellence “Nanosystems Initiative Munich” (NIM).
The MST technology is based on the principle of thermophoresis, a physical phenomenon that causes directed movement of particles along a temperature gradient. The method involves setting up a short-range temperature gradient by irradiating a liquid sample with an infrared laser, and monitoring the resulting change in the distribution of a labelled molecule of interest by fluorescence microscopy. If the labelled molecules bind to other components in the solution, their thermophoretic behavior is altered, and the change in their rate of migration allows one to measure the strength of the interaction.
Thermophoresis is exquisitely sensitive to changes in many of the most basic properties of biomolecules, including surface parameters such as electric charge and especially the hydration sphere, that is affected by all intermolecular binding reactions. Unlike conventional methods, the MST technique is capable of detecting interactions between partners that differ widely in their dimensions and masses, as is the case when ions or small molecules bind to proteins. So the new procedure will make it possible to study interactions that have hitherto eluded quantitative investigation.
One further advantage of the process is its unrivalled simplicity. The analysis is carried out in solution, which saves time and avoids the need to immobilize either of the binding partners, a step that might otherwise distort the results. Furthermore, binding takes place under near-physiological conditions, and therefore provides both basic and clinical researchers with biologically relevant data that can lead to new mechanistic insights and accelerate the development of effective medicinal compounds.
“The MST technology opens up new opportunities in drug development, especially in the field of small-molecule pharmaceuticals“, says Dr. Stefan Duhr, Managing Director of NanoTemper. For example, the method can rapidly determine whether promising drug candidates bind to components in the blood, a phenomenon that may prevent them from reaching their site of action in physiologically relevant concentrations. “At present it is very difficult to obtain this kind of data before a complex and costly clinical study is conducted“, says Duhr.
Source: LMU Munich /...
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