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

生物大分子(蛋白质、DNA、RNA)的静态结构决定了它们的功能，但在生理条件下，结构的变化(结构动力学)在生物机制中同样重要。这意味着，为了设计与大的、灵活的生物分子结合的分子或影响它们在溶液中采用的构象的分子，我们必须获得关于目标分子的准确的结构和动态信息。目前的分析方法分为两类，一类是提供详细结构的方法(X射线结晶学、低温电子显微镜、蛋白质观察的核磁共振)，但应用起来很耗时(低通量)；另一类是快速报告分子间相互作用，但提供的结构洞察力很少，如配体观察的核磁共振、自然质谱学或表面等离子体共振。二维红外(2D-IR)光谱学使我们对结构和动态信息的使用发生了阶段性的变化，它使用一系列中红外激光脉冲来激发分子振动，并生成生物分子的3D结构、结构动力学和分子间相互作用的独特2D‘图谱’。至关重要的是，现代激光技术大大缩短了获取2D-IR光谱所需的时间，为2D-IR用作高通量的基于结构的筛选工具或实时探测复杂和不断演变的分子混合物开辟了令人兴奋的可能性。最近，由York领导的世界领先的研究开发了对水(H2O)和生物液中生物分子的结构和动力学的2D-IR测量。这项发明消除了在红外测量之前用重水(D2O)代替水的传统需要，这既耗时又昂贵。此外，这一新能力为无需样品干燥的生物体液无标记分子分析铺平了道路(Chem Sci，10,6448-6456,2019，编辑之选)和在水中进行2D-IR蛋白质药物筛选实验。我们相信，在生理相关条件下对分子进行基于结构/动力学的快速2D-IR分析将填补我们分析能力的重要空白，改变生物化学研究，并为医疗诊断提供新的工具。为了挖掘这一巨大的潜力，我们建议在约克建造一台全球独一无二的高通量2D-IR仪器，该仪器可以在一分钟内测量微升体积的样品。这一新能力将：1)通过量化用于疾病诊断的生物液的生物分子含量来促进生物医学诊断，而无需标记、干燥或使用抗体。2)通过实现基于结构的生物液中传感器-分析物相互作用的优化，增强下一代光子生物传感器。3)通过对与蛋白质和核酸结合的候选分子的分子合成和基于结构的筛选的实时机械洞察，提供用于化学生物学和药物设计的使能技术，而无需昂贵、费力地将H2O替换为D2O。4)首次测定了生物分子和配体在天然溶剂中的结构动力学。

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.