High throughput 2D-IR analysis of biomolecules under physiological conditions

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The static structure of biomacromolecules (proteins, DNA, RNA) defines their function but, under physiological conditions, changes in structure (structural dynamics) are equally important in biological mechanisms. This means that, in order to design molecules that bind to large, flexible biomolecules or which influence the conformations that they adopt in solution, we must have access to accurate structural and dynamic information about the target molecule.Current analytical methods fall into two categories, those that provide detailed structures (X-ray crystallography, cryo-EM, protein-observed NMR), but are time consuming to apply (low throughput) and those that report rapidly on intermolecular interactions but provide little structural insight such as ligand-observed NMR, native mass spectrometry or surface plasmon resonance. A step change in our use of structural and dynamic information is offered by two-dimensional infrared (2D-IR) spectroscopy, which uses a sequence of mid-IR laser pulses to excite molecular vibrations and generate a unique 2D 'map' of the 3D structure, structural dynamics and intermolecular interactions of biological molecules. Crucially, modern laser technology has dramatically shortened the amount of time needed to acquire a 2D-IR spectrum, opening up exciting possibilities for 2D-IR to be used as a high-throughput structure-based screening tool or to probe complex and evolving molecular mixtures in real time. Recently, world-leading research led by York has developed 2D-IR measurements of the structure and dynamics of biological molecules in water (H2O) and biofluids. This invention removes the traditional need for replacement of water with 'heavy water' (D2O) before IR measurements, which is both time consuming and expensive. Moreover, this new ability paves the way to label-free molecular analysis of biofluids without sample drying (Chem Sci, 10, 6448-6456, 2019, Editors' Choice) and 2D-IR protein-drug screening experiments in H2O.We believe that rapid structure/dynamics-based 2D-IR analysis of molecules under physiologically relevant conditions will fill an important gap in our analytical capability, transforming biological chemistry research and providing a new tool for healthcare diagnostics. To exploit this enormous potential, we propose to build a globally unique high throughput 2D-IR instrument at York that can measure microlitre volume samples in under a minute. This new capability will: 1) Advance biomedical diagnostics by quantifying the biomolecular content of biofluids for disease diagnosis without labelling, drying or use of antibodies.2) Enhance next-generation photonic biosensors by enabling structure-based optimisation of sensor-analyte interactions in biofluids. 3) Deliver enabling technology for chemical biology and drug design via real-time mechanistic insight into molecular synthesis and structure-based screening of candidate molecules binding to proteins and nucleic acids without expensive, laborious replacement of H2O with D2O. 4) Measure structural dynamics of biomolecules and ligands in their native solvent for the first time.