Developing and applying genetically encoded proteins as pre-resonant coherent Raman scattering tags for next-generation live-cell imaging.

该研究项目将通过利用合成生物学设计和应用基于拉曼的微光谱的蛋白质标签来开发下一代生物成像方法。光学显微镜是理解细胞内生物过程不可或缺的工具，也是目前在活细胞和组织内获得高空间和时间分辨率的唯一实用手段。荧光显微镜提供了一种高度敏感和特异性的生物分子可视化方法，并彻底改变了细胞成像，特别是在荧光蛋白（FPs）的发现之后，荧光蛋白能够在生命系统内对特定目标进行遗传标记。然而，也存在缺陷，因为荧光探针容易发生光漂白和相关的细胞毒性，这阻碍了它们的使用，特别是在长时间尺度的活细胞成像中，这对理解生物过程和潜在疾病状态至关重要。此外，这些探针的发射光谱相当宽，限制了同时监测多个目标。本项目将寻求将遗传编码荧光蛋白的优势与振动拉曼标签的优势结合起来，开发一种新的活细胞成像方法。潜在的想法是在FP功能中心附近的特定位置结合通常不存在于生物学中的拉曼活性化学键。这些新的基因编码成像探针将允许您利用称为电子预共振拉曼散射（PRRS）的过程，将拉曼信号增强约2-4个数量级，因此可以对少量这些标签进行成像。蛋白质工程和重编程遗传密码方法将被用于将含有拉曼活性化学键的非天然氨基酸整合到通过硅内设计选择的FPs的最佳位置。使用先进的拉曼散射微光谱学，您将量化和了解您设计的蛋白质中的PRRS，并将其应用于图像细胞事件。最初，您将生成微管蛋白fp结构来演示静态（微管）和动态（组装/拆卸）细胞结构的技术。至关重要的是，在光稳定性方面，PRRS有望超越传统的荧光方法，您将通过整个细胞周期的长期成像来验证这一特性。这将使我们能够更好地理解细胞生命周期中管蛋白组装/拆卸的动力学，以及抗癌和抗炎药物如何影响这些过程。

This research project will develop next generation biological imaging methods by using synthetic biology to design and apply protein tags for Raman-based micro-spectroscopy. Optical microscopy is an indispensable tool that is pivotal to understanding biological processes in the cell and is currently the only practical means of obtaining high spatial and temporal resolution within living cells and tissues. Fluorescence microscopy has provided a highly sensitive and specific method of visualizing biomolecules and has revolutionised cell imaging, especially after the discovery of fluorescent proteins (FPs) that enabled genetic tagging of specific targets inside living systems. However, there are flaws, as fluorescent probes are prone to photo-bleaching and associated cytotoxicity which hamper their use, especially for imaging over long timescales in live cells which is critical to understanding biological process and underlying disease states. Moreover, the emission spectrum of these probes is quite broad, limiting simultaneous monitoring of multiple targets.This project will seek to merge the advantages of genetically encoded fluorescent proteins with those of vibrational Raman tags to develop a new live cell imaging approach. The underlying idea is to incorporate Raman-active chemical bonds not normally present in biology at specific locations near the functional centre of a FP. These new genetically encoded imaging probes will allow you to exploit a process called electronic pre-resonant Raman scattering (PRRS), enhancing the Raman signal by some 2-4 orders of magnitude, and therefore enabling imaging of a small number of these tags. Protein engineering together with a reprogrammed genetic code method will be used to incorporate non-natural amino acids containing Raman active chemical bonds at optimal positions in selected FPs, selected via in-silico design. Using advanced Raman scattering micro-spectroscopy you will quantify and understand PRRS in your designed proteins and apply them to image cellular events. Initially, you will generate tubulin-FP constructs to demonstrate the technique on static (microtubules) and dynamic (assembly/disassembly) cellular structures. Crucially, PRRS is expected to surpass traditional fluorescence approaches in terms of photo-stability, and you will verify this property by long term imaging throughout the cell cycle. This will in turn allow us to better understand the dynamics of tublin assembly/disassembly throughout the life time of a cell, and how anticancer and anti-inflammatory drugs impact on these processes.