Developing genetically encoded ultra-correlative tags for functional studies employing light and electron microscopy

一个关键的生物医学目标是了解细胞器和蛋白质如何合作，使器官在“正常”和患病条件下工作，最终目的是改善我们的健康。然而，目前探索这些结构-功能关系的方法具有许多缺点。在这些限制中，最重要的是由标准电子显微镜方法设定的分辨率限制以及与跨组织和分子尺度长度的功能和结构数据相关的困难。我们建议开发一种基因编码的超相关标签，用于标记基因的多重结构和功能分析。我们将开发基于电子显微镜可检测的重金属结合肽的新型遗传标签。这种方法将使蛋白质定位精度比现有方法高一个数量级，并减轻了与经典电子显微镜方法相关的许多缺点。此外，我们将联合收割机与已建立的光学显微镜方法相结合，以创建一个“双标签”，使相关的光学和电子显微镜（CLEM）结构评估以及活细胞功能成像相同的基因。最后，通过使用基因编辑技术整合这种新的功能性CLEM遗传标记策略，我们将实现真正相关的分子水平分辨率，并提供定量结构-功能信息。重要的是，该方法不限于研究单一靶蛋白的结构-功能关系，事实上，使用广泛的相容性共价标记标签和特异性金属结合肽，可以同时精确评估多种蛋白质。所提出的方法可容易地适用于跨越单细胞系统到整个生物体生理学的不同生物和疾病相关系统。

A key biomedical goal is to understand how organelles and proteins cooperate to make organs work, in both 'normal' and diseased conditions, with the ultimate aim to improve our health. However, current methods to explore these structure-function relationships have a number of drawbacks. Not least amongst these limitations are the resolution limit set by standard electron microscopy approaches and the difficulty associated with correlating functional and structural data across tissue and molecular scale length. We propose to develop a genetically encoded ultra-correlative tag for multiplexing structural and functional analysis of labelled genes. We will develop novel genetic tags based on heavy metal binding peptides detectable with electron microscopy. This method will give protein localisation precision an order of magnitude greater than established methods and mitigate many of the drawbacks associated with classic electron microscopy approaches. Further, we will combine it with established light microscopy methods to create a 'dual tag' that enables both correlative light and electron microscopy (CLEM) structural assessments as well as live cell functional imaging on the same genes. Finally, by integrating this new functional CLEM genetic tagging strategy using gene editing techniques we will achieve truly correlative molecular level resolution and provide quantitative structure-function information. Importantly, this method is not limited to study the structure-function relationship of a single target protein, in fact using a wide array of compatible covalent labelling tags and specific metal binding peptides, multiple proteins can be simultaneously assessed with precision. The proposed approaches are readily applicable across different biological and disease relevant systems spanning single cell systems through to whole organism physiology.