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.

氟原子几乎从未在生命的自然分子中发现，例如：肽、蛋白质、核酸和酶辅助因子。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灯塔，利用 19F NMR 研究它们的生化转变、与其他生物分子的相互作用以及它们的形状变化。 NMR 是一种提供有关分子或分子部分存在的化学环境信息的技术，19F NMR 对于生物学来说是一种特别有用的工具，因为每种不同的含氟分子在光谱的不同部分提供独特且可测量的信号，这意味着我们可以进行实时实验，同时测量复杂混合物、不常见溶剂（例如溶剂）中的多种物质。生物体液和细胞内。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，从而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的灵敏度相对较低。然而，这可以通过以下方式克服：i) 减少干扰背景信号，ii) 使用提高信噪比的 NMR 冷冻探针，以及 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，我们的目标是使用购买的专用低温探针和在爱丁堡开发的新 SHARPER 方法，将氟化生物分子检测的灵敏度相对于室温探针显着提高一百倍以上，作为变革性的中心支柱生物系统研究。这项研究的一个方面是开发新工具，将氟化“标签”附加到 DNA 和蛋白质（包括患者来源的样本）上，从而利用新型冷冻探针和 SHARPER 方法的卓越灵敏度。当没有其他 19F 信号时，将氟化报告基因连接到生物分子上进行 19F NMR，使我们能够清楚地观察和测量低微摩尔至纳摩尔浓度下不同 DNA 物种之间由于信号的独特变化而发生的相互作用。这也将用于在蛋白质折叠和聚集过程中使用荧光方法测量存在但无法观察到的蛋白质的不同瞬时形式。我们还将使用氟化笼分子的独特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理解生活规则。鉴于 19F NMR 的上述优点，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型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 19F NMR 通过观察氟化酶底物向新产品的转化，了解如何利用生物技术实现合成原料的可持续获取 - 重要的是提供使用其他方法无法轻松观察到的短寿命中间体物种的结构信息方法。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。我们将扩展生物合成工具箱，开发绿色人工酶，可以在温和、安全的条件下将氟原子安装到新的化学构件中，这将彻底改变氟化分子的制备。总体而言，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 19F NMR 作为研究动态生物过程的工具的未开发潜力。