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

生物医学的一个关键目标是了解细胞器和蛋白质在“正常”和患病情况下如何合作使器官工作，最终目的是改善我们的健康。然而，目前探索这些结构-功能关系的方法有许多缺点。这些限制中最重要的是标准电子显微镜方法设定的分辨率限制，以及跨组织和分子尺度长度关联功能和结构数据的困难。我们建议开发一种遗传编码的超相关标签，用于标记基因的多路结构和功能分析。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。这种方法将给蛋白质定位精度一个数量级大于既定的方法，并减轻了许多与经典电子显微镜方法相关的缺点。此外，我们将把它与现有的光学显微镜方法结合起来，创建一个“双标签”，使相关的光学和电子显微镜（CLEM）结构评估以及对相同基因的活细胞功能成像成为可能。最后，通过将这种新的功能CLEM遗传标记策略与基因编辑技术相结合，我们将实现真正相关的分子水平分辨率，并提供定量的结构-功能信息。重要的是，该方法并不局限于研究单个靶蛋白的结构-功能关系，事实上，使用一系列兼容的共价标记标签和特定的金属结合肽，可以同时精确地评估多个蛋白质。所提出的方法很容易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.