Supporting 19F-centered NMR investigations across a range of biological applications

支持一系列生物应用中以 19F 为中心的 NMR 研究

• 批准号: BB/X019756/1
• 负责人: Dusan Uhrin
• 金额: $ 28.37万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX019756%2F1
• 关键词: Supporting; 19F; centered; NMR; investigations

The fluorine atom is almost never found in the natural molecules of life e.g. peptides, proteins, nucleic acids and enzyme cofactors. Therefore, carefully installing fluorine atoms into these biomolecules can provide a beacon with which to study their biochemical transformations, interactions with other biomolecules and changes in their shape using 19F NMR. NMR is a technique that provides information about the chemical environment in which a molecule or part of a molecule exists and 19F NMR is a particularly useful tool for biology because each different fluorine-containing molecule provides a distinct and measurable signal in different parts of the spectrum, meaning that we can perform real-time experiments simultaneously measuring multiple species in complex mixtures, unusual solvents e.g. biological fluids and in cells. Moreover, the absence of natural fluorine means that we can selectively observe only the events involving fluorinated biomolecules, providing a clear window into an otherwise very complex and crowded world at the molecular scale. One of the challenges in using NMR for biology has been the relatively low sensitivity of traditional methods. However, this can be overcome by i) reducing interfering background signals, ii) using NMR cryoprobes that improve signal-to-noise ratios, and iii) using novel methods to amplify the signal, finally allowing us to study dilute samples in biological environments. In this project, we aim to use the purchase of a dedicated cryo probe and new SHARPER methods developed at Edinburgh to significantly enhance the sensitivity for detection of fluorinated biomolecules by more than hundred-fold relative to the room temperature probes as a transformative central pillar for studies on biological systems. One aspect of this research that will exploit the exceptional sensitivity of the new cryoprobe and SHARPER methods is the development of new tools to attach fluorinated 'tags' to DNA and proteins, including patient-derived samples. Attaching a fluorinated reporter to a biomolecule for 19F NMR when there are no other 19F signals, allows us to clearly observe and measure the interactions between different DNA species at low micromolar-to-nanomolar concentrations due to distinctive changes in the signals. This will be used also to measure the different transient forms of proteins that exist, but cannot be observed, using fluorescence methods during protein folding and aggregation. We will also use the distinct and quantitative signals for fluorinated cages molecules to understand how tightly they bind to blood proteins. These outcomes will allow us to better understand the rules of life. Given the above benefits of 19F NMR, this is also an outstanding method to study biochemical reactions and transformations in real-time. This will be used to conveniently measure the biological reactivity and stability of new fluorinated Raman imaging tags and enzyme probes in whole cells and in cellular fluids. We will also use 19F NMR to understand how to harness biotechnology for the benefit of sustainable access to synthetic feedstocks by observing the transformation of fluorinated enzyme substrates into new products - importantly providing structural information on short-lived intermediate species that cannot be observed easily using other methods. The preparation of fluorinated molecules can be challenging and often requires the use of dangerous fluorine gas and complex apparatus. We will expand the biosynthetic toolbox to develop green artificial enzymes that can install fluorine atoms into new chemical building blocks under mild and safe conditions, which will revolutionise the preparation of fluorinated molecules. Overall, access to a dedicated cryoprobe, coupled with new analysis methods and synthetic fluorinated tools will release the untapped potential of 19F NMR as a tool with which to study dynamic biological processes.

在生命的自然分子中，几乎找不到氟原子，例如肽，蛋白质，核酸和酶辅因子。因此，小心地将氟原子安装到这些生物分子中可以提供一个信标，以研究其生化转化，与其他生物分子的相互作用以及使用19F NMR的形状变化。 NMR is a technique that provides information about the chemical environment in which a molecule or part of a molecule exists and 19F NMR is a particularly useful tool for biology because each different fluorine-containing molecule provides a distinct and measurable signal in different parts of the spectrum, meaning that we can perform real-time experiments simultaneously measuring multiple species in complex mixtures, unusual solvents e.g.生物流体和细胞中。此外，缺乏天然氟意味着我们只能选择性地观察涉及氟化生物分子的事件，从而在分子尺度上为一个非常复杂且拥挤的世界提供了一个清晰的窗口。将NMR用于生物学的挑战之一是传统方法的敏感性相对较低。但是，这可以通过i）减少干扰背景信号，ii）使用改善信号噪声比率的NMR冷冻探针，以及使用新型方法扩增信号，最终使我们能够在生物学环境中研究稀释样品。在该项目中，我们旨在使用在爱丁堡开发的专用冷冻探针和新的更清晰的方法，以显着提高对氟化生物分子检测的敏感性，相对于室温探测器作为生物学系统研究的一种变革性的中心支柱，相对于室温探测器作为一种变革性的中心支柱。这项研究的一个方面将利用新的冷冻探针和更清晰的方法的特殊灵敏度的是开发新工具，将氟化的“标签”附加到DNA和蛋白质（包括患者衍生的样品）上。在没有其他19F信号时，将氟化的记者连接到19f NMR的生物分子上，使我们能够清楚地观察并测量由于信号的独特变化而导致的低微摩尔到纳米尔浓度下不同DNA物种之间的相互作用。这也将用于测量存在蛋白质折叠和聚集期间荧光方法存在但无法观察到的蛋白质的不同瞬态形式。我们还将为氟化的笼子分子使用独特的定量信号，以了解它们与血液蛋白的结合程度。这些结果将使我们能够更好地理解生活规则。鉴于19F NMR的上述好处，这也是实时研究生化反应和转化的出色方法。这将用于方便地测量全细胞和细胞液中新的氟拉曼成像标签和酶探针的生物反应性和稳定性。我们还将使用19F NMR来了解如何利用生物技术来利用可持续的合成原料，通过观察氟化酶底物为新产品的转化 - 重要的是，重要的是，使用其他方法可以轻松地观察到短暂的中间物种的结构信息。氟化分子的制备可能具有挑战性，通常需要使用危险的氟气体和复杂的设备。我们将扩展生物合成工具箱，以开发绿色的人造酶，这些酶可以在轻度和安全的条件下将氟原子安装到新的化学构建块中，这将彻底改变氟化分子的制备。总体而言，使用专用的冷冻探针，再加上新的分析方法和合成氟化工具，将释放19F NMR的未开发潜力，作为研究动态生物学过程的工具。