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.

氟原子几乎从未在天然生命分子中发现，例如肽、蛋白质、核酸和酶辅因子。因此，仔细地将氟原子安装到这些生物分子中可以提供一个信标，使用19 F NMR研究它们的生化转化，与其他生物分子的相互作用以及它们的形状变化。NMR是一种提供分子或分子部分存在的化学环境信息的技术，19 F NMR是生物学中特别有用的工具，因为每个不同的含氟分子在光谱的不同部分提供了不同的可测量信号，这意味着我们可以进行实时实验，同时测量复杂混合物中的多个物种，不寻常的溶剂，例如生物液体和细胞中。此外，天然氟的缺乏意味着我们只能选择性地观察涉及氟化生物分子的事件，为分子尺度上非常复杂和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相对较低的灵敏度。然而，这可以通过以下方式克服：i）减少干扰背景信号，ii）使用NMR冷冻探针提高信噪比，iii）使用新方法放大信号，最终使我们能够研究生物环境中的稀释样品。在这个项目中，我们的目标是使用在爱丁堡开发的专用冷冻探针和新的SHARPER方法，以显着提高检测氟化生物分子的灵敏度，相对于室温探针超过100倍，作为生物系统研究的变革性中心支柱。这项研究的一个方面将利用新的冷冻探针和SHARPER方法的特殊灵敏度，开发新的工具来将氟化“标签”附着在DNA和蛋白质上，包括患者来源的样本。当没有其他19 F信号时，将氟化报告分子连接到19 F NMR的生物分子上，使我们能够清楚地观察和测量由于信号的独特变化而导致的低微摩尔至纳摩尔浓度下不同DNA物质之间的相互作用。这也将用于测量蛋白质折叠和聚集过程中使用荧光方法存在但无法观察到的不同瞬时形式的蛋白质。我们还将使用氟化笼分子的独特和定量信号来了解它们与血液蛋白结合的紧密程度。这些结果将使我们更好地了解生活的规则。鉴于19 F NMR的上述优点，这也是实时研究生化反应和转化的杰出方法。这将用于方便地测量全细胞和细胞液中新的氟化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19 F NMR来了解如何利用生物技术，通过观察氟化酶底物转化为新产品来可持续地获得合成原料-重要的是提供使用其他方法无法轻易观察到的短寿命中间物种的结构信息。氟化分子的制备可能具有挑战性，并且通常需要使用危险的氟气和复杂的设备。我们将扩大生物合成工具箱，开发出绿色人造酶，可以在温和安全的条件下将氟原子安装到新的化学构建单元中，这将彻底改变氟化分子的制备。总的来说，使用专用冷冻探针，再加上新的分析方法和合成氟化工具，将释放19 F NMR作为研究动态生物过程的工具的未开发潜力。