A universal pipeline for genetically-encoded fluorescent biosensor production

高分辨率生物成像的进步使生命科学和医学的研究发生了革命性的变化，使活细胞内蛋白质的定位和运动得以可视化和量化。然而，除了蛋白质，还有许多其他重要的生物分子，如代谢物和信号分子，并没有被证明是那么容易以同样的方式量化。问题是，人们必须为每个生物分子设计一种荧光传感器蛋白质。当这些蛋白质与目标分子结合时，它们的荧光性质会改变，从而提供细胞内其浓度的读数。这些荧光传感器很难制造，部分原因是它们依赖于识别合适的蛋白质，该蛋白质既能与靶分子结合，又能在这样做的基础上显著改变形状-正是这种形状变化导致连接的荧光蛋白(如绿色荧光蛋白(GFP))的性质发生可检测的变化。我们设计了一种构建荧光传感器蛋白的替代方法，该方法不需要受体蛋白经历较大的形状变化。这大大增加了潜在有用的受体蛋白的范围，也允许从抗体库中识别受体蛋白。这项提议是通过制造两个测试传感器来评估新的传感器设计，一个用于多肽，另一个用于激素雌激素。如果成功，我们的设计将代表一个通用平台，任何分子的生物传感器都可以在这个平台上构建。我们将向学术研究人员提供这个平台，并预计它将极大地加快收集关于生物体内小分子动力学的至关重要的数据。

Advances in high-resolution bio-imaging have revolutionised research in the life sciences and medicine allowing the localisation and movement of proteins within living cells to be visualised and quantified. However, in addition to proteins, there are many other important biomolecules such as metabolites and signal molecules which have not proved so easy to quantify in the same way. The problem is that one has to design a fluorescent sensor protein for each biomolecule. The fluorescence properties of these proteins change when they bind the target molecule, providing a readout of its concentration in the cell. These fluorescent sensors are difficult to make, partly because they rely on identifying a suitable protein that both binds the target molecule and, upon doing so, changes shape substantially - it is this shape change that causes the detectable change in properties of a linked fluorescent protein such as green fluorescent protein (GFP).We have devised an alternative method of constructing fluorescent sensor proteins that does not require the receptor protein to undergo a large shape-change. This massively increases the range of potentially useful receptor proteins and also allows receptor proteins to be identified from antibody libraries. This proposal is to assess the new sensor design by making two test sensors, one for a peptide and one for the hormone estrogen. If successful, our design would represent a universal platform from which biosensors for any molecule could be constructed. We would make this platform available to academic researchers and anticipate that it would dramatically speed up the collecting of critically important data about small molecule dynamics in living organisms.