High Resolution Imaging Using Transient Binders

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To better understand the behaviour of any disease and to develop treatment options for it, we need to understand processes occurring on the single molecule level as well as the cellular and tissue levels. However, biomolecules are highly dynamic and have complex structures which are difficult to observe simultaneously. Biomolecules such as DNA and proteins interact with many other molecules to perform their biological function. Exploiting these interactions with molecules such as drugs, to block or enhance function can be used to treat or prevent diseases. However, few experimental techniques can capture this information at the relevant spatiotemporal scales. This proposal will develop Localization Atomic Force Microscopy (LAFM) methods to enable near atomic resolution imaging of single biomolecules interacting with binding partners. By localizing the height signals as small molecules transiently bind to DNA or proteins, the resolution of the LAFM method will be increased and enable imaging of where drugs bind, how long they bind for and their effects on how the biomolecule functions. Additionally, the combined development of automatic spectroscopy methods will enable the measurement of binding kinetics on the microsecond to seconds timescale. This combination of high spatial and temporal resolution will allow new insight into protein and DNA including drug interactions, molecular mechanisms of disease and surface properties, opening opportunities to identify new drug molecules or targets.Technologies such as X-ray crystallography, surface plasmon resonance, isothermal titration calorimetry and more recently, cryo-electron microscopy and artificial intelligence have proven to be invaluable in understanding drug interactions with biomolecules in atomic detail. However, these technologies currently only provide static structures and ensemble averages of the most stable, lowest energy states across many molecules. To perform functions, proteins must undergo rapid dynamic changes; something which only a few techniques can currently capture across specific timescales with some constraints on protein size and order. My previous research has demonstrated the ability to obtain 4Å spatial resolution on single molecules with LAFM and a 10 microsecond time resolution using single point measurements with High-Speed AFM Height Spectroscopy. This, combined with my expertise in biophysics and high-speed AFM, will allow me to advance these techniques to further increase resolution and achieve quantitative binding kinetics with new LAFM methods.To develop my methodology, specifically designed DNA structures will be used as benchmark systems with fully predetermined binding site locations. A variety of target sites and binding molecules of varying size, affinity and binding mechanism will be explored to optimize localization algorithms, imaging parameters and conditions. These experiments will determine general rules governing resolution gains, localization precision at specific sites and the limits of detection for different binding interactions. Accurate and automated tip positioning at binding sites with predictive drift control will be developed for 10,000-fold increased time resolution for rapid binding kinetics to be measured using Height Spectroscopy. These newly developed techniques will then be used to image and measure kinetics of sugars to carbohydrate binding proteins often used by viruses, bacteria and other pathogens. Binding of drug fragments, peptides, and small proteins to 3 different proteins will be studied by the new technology to detect drug locations, kinetics and the resulting protein structural changes. In parallel with these experiments this fellowship will allow me to develop my leadership and research management skills, open-source software, and links to industry and academia, to ensure that the potential of these new methodologies are fully exploited.