High Resolution Imaging Using Transient Binders

为了更好地了解任何疾病的行为并为其开发治疗方案，我们需要了解单分子水平以及细胞和组织水平上发生的过程。然而，生物分子是高度动态的，并且具有难以同时观察的复杂结构。生物分子如DNA和蛋白质与许多其他分子相互作用以执行其生物功能。利用这些与药物等分子的相互作用来阻断或增强功能，可用于治疗或预防疾病。然而，很少有实验技术可以捕捉到这些信息在相关的时空尺度。该提案将开发定位原子力显微镜（LAFM）方法，以实现与结合伙伴相互作用的单个生物分子的近原子分辨率成像。通过将高度信号定位为小分子与DNA或蛋白质瞬时结合，LAFM方法的分辨率将增加，并能够成像药物结合的位置，结合时间以及它们对生物分子功能的影响。此外，自动光谱学方法的联合开发将使结合动力学的测量在微秒到秒的时间尺度上。这种高空间和时间分辨率的结合将允许对蛋白质和DNA的新见解，包括药物相互作用、疾病的分子机制和表面性质，从而为识别新的药物分子或靶点提供机会。技术如X射线晶体学、表面等离子体共振、等温滴定量热法以及最近的技术，冷冻电子显微镜和人工智能已被证明在了解药物与生物分子的原子细节相互作用方面是非常宝贵的。然而，这些技术目前仅提供许多分子中最稳定、最低能量状态的静态结构和系综平均值。为了发挥功能，蛋白质必须经历快速的动态变化;目前只有少数技术可以在特定的时间尺度上捕获蛋白质的大小和顺序。我以前的研究已经证明了LAFM在单分子上获得4 μ s空间分辨率的能力，以及使用高速AFM高度光谱仪进行单点测量获得10微秒时间分辨率的能力。这一点，结合我在生物物理学和高速AFM的专业知识，将使我能够推进这些技术，以进一步提高分辨率，并实现定量结合动力学与新的LAFM methods.To发展我的方法，专门设计的DNA结构将被用作基准系统与完全预定的结合位点的位置。将探索不同大小、亲和力和结合机制的各种靶位点和结合分子，以优化定位算法、成像参数和条件。这些实验将确定控制分辨率增益、特定位点的定位精度和不同结合相互作用的检测限的一般规则。将开发具有预测漂移控制的结合位点处的准确和自动化尖端定位，以使时间分辨率提高10，000倍，从而使用高度光谱法测量快速结合动力学。然后，这些新开发的技术将用于成像和测量糖与病毒、细菌和其他病原体经常使用的碳水化合物结合蛋白的动力学。将通过新技术研究药物片段、肽和小蛋白与3种不同蛋白质的结合，以检测药物位置、动力学和由此产生的蛋白质结构变化。在这些实验的同时，这项奖学金将使我能够发展我的领导和研究管理技能，开源软件，以及与工业界和学术界的联系，以确保这些新方法的潜力得到充分利用。

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.