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

开发和应用基因编码蛋白质作为下一代活细胞成像的预共振相干拉曼散射标签。

• 批准号: 2434545
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2434545
• 关键词: Developing; applying; genetically; encoded; proteins

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.

该研究项目将通过使用合成生物学设计和应用蛋白质标签来开发下一代生物成像方法，以用于基于拉曼的微光谱法。光学显微镜是一种必不可少的工具，对于理解细胞中的生物学过程至关重要，目前是在活细胞和组织中获得高空间和时间分辨率的唯一实际手段。荧光显微镜提供了一种可视化生物分子的高度敏感和特定的方法，并彻底改变了细胞成像，尤其是在发现荧光蛋白（FPS）之后，它启用了生命系统中特定靶标的基因标记。但是，存在缺陷，因为荧光探针容易出现造影和相关的细胞毒性，这阻碍了它们的使用，尤其是在活细胞中长时间尺度上成像，这对于理解生物学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广泛，同时监测了多个目标。该项目将寻求将遗传编码的荧光蛋白与振动拉曼标签的优势合并，以开发一种新的活细胞成像方法。潜在的想法是结合通常在FP功能中心附近的特定位置中通常存在于生物学中的拉曼活性化学键。这些新的遗传编码的成像探针将使您能够利用称为电子前谐波拉曼散射（PRR）的过程，从大约2-4个数量级增强了拉曼信号，因此可以对这些标签进行成像。蛋白质工程与重编程的遗传密码方法将用于合并含有在选定FPS中最佳位置的拉曼活性化学键的非天然氨基酸，这是通过Silico设计选择的。使用先进的拉曼散射微光谱镜检查，您将量化和理解设计蛋白质中的PRR，并将其应用于图像蜂窝事件。最初，您将生成小管蛋白-FP构建体，以演示静态（微管）和动态（装配/拆卸）细胞结构上的技术。至关重要的是，PRR有望在光稳定性方面超过传统的荧光方法，并且您将在整个细胞周期中通过长期成像来验证此属性。反过来，这将使我们能够更好地理解Tublin组装/拆卸的动力学在整个细胞的生命期，以及抗癌和抗炎药如何对这些过程的影响。