Microscale thermophoresis for assessing diverse molecular interactions.

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Molecular interactions underlie the life and death signals of all cells. There is a large diversity in the nature of the interacting molecules controlling these processes. For example, binding of single ions can radically alter the conformation and function of small proteins. In contrast, translation machinery is made up of dozens of ribosomal proteins, nucleic acids and ions, forming an enormous 4.3 MDa complex in higher eukaryotes. There is also a wide variability in the strengths of these interactions, which typically range from ~nM to mM affinity in nature. ******Measuring molecular interaction affinities provides insights on the energies, relative probabilities and lifetimes, central to developing new research tools and drugs as well as bioprocess optimization in industry. Numerous approaches and specialized equipment have been engineered to characterize molecular interactions such as isothermal titration calorimetry (ITC), surface plasmon resonance spectroscopy (SPR), analytical ultracentrifugation (AUC) and nuclear magnetic resonance (NMR) spectroscopy. However, these techniques have clear shortcomings when considering the diverse affinities and interaction types in nature. ITC and AUC require pure samples at relatively high concentrations; SPR demands sample immobilization; NMR uses large amounts of sample. Additionally, these approaches are technically demanding, limiting their use by researchers and trainees with varied expertise. Finally, many of these approaches are partial in the strengths of the interactions detected, typically measuring sub-mM affinities. ******This multi-applicant research proposal aims to acquire funds to purchase a microscale thermophoresis (MST) unit, which can overcome these limitations and be used to meet the goals of several NSERC-funded research programs at Western. Remarkably, MST can measure affinities ranging from ~nM mM, assess interactions in complex heterogeneous mixtures and detect interactions ranging from small ions to enormous complexes. Further, the system requires only miniscule volumes (~4 microL) and sample amounts (~pmol), is rapid (~minutes) and is technically straightforward, making it widely accessible to multiple disciplines. Currently, there are no MST units available at Western, highlighting an urgent need to enhance our capabilities in assessing diverse molecular interactions. ******The proposed MST unit will immediately benefit the 6 NSERC-funded research programs of the applicants. Nevertheless, the instrument will also be available for use by individuals in the Faculty of Science, Engineering and Schulich School of Medicine and Dentistry. Ultimately, trainees from diverse fields will be exposed to this modern instrumentation that is simple to use and provides essential data for understanding the respective systems under study. Thus, the acquisition of an MST unit will provide both tangible research and training benefits with applicability in industry, academia and health-related fields.