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

氟原子几乎不存在于生命的天然分子中，例如多肽、蛋白质、核酸和酶辅助因子。因此，仔细地在这些生物分子中安装氟原子可以为利用19F核磁共振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提供一个灯塔。核磁共振是一种提供有关分子或分子部分存在的化学环境的信息的技术，而19F核磁共振对于生物学来说是一个特别有用的工具，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，这意味着我们可以同时进行实时实验，测量复杂混合物、特殊溶剂(如生物液和细胞)中的多种物种。此外，天然氟的缺乏意味着我们只能选择性地观察到涉及含氟生物分子的事件，这为我们在分子尺度上了解一个原本非常复杂和拥挤的世界提供了一个清晰的窗口。将核磁共振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i)减少干扰背景信号，ii)使用可提高信噪比的核磁共振低温探头，以及iii)使用新的方法放大信号，最终允许我们在生物环境中研究稀释样品来克服。在这个项目中，我们的目标是使用购买的专用低温探头和爱丁堡开发的新的更锐利的方法，显著提高检测氟化生物分子的灵敏度，比常温探头高出数百倍以上，成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头和更锐利的方法的特殊敏感性，就是开发新的工具，将氟化的“标签”附加到DNA和蛋白质上，包括患者提取的样本。在没有其他19F信号的情况下，将一个氟化报告器连接到生物分子上进行19F核磁共振，使我们能够在低微摩尔到纳摩尔浓度下清楚地观察和测量不同DNA物种之间的相互作用，这是由于信号的显著变化。这也将被用来测量存在但不能观察到的蛋白质的不同瞬时形式，在蛋白质折叠和聚集过程中使用荧光方法。我们还将使用氟化笼子分子的不同和定量信号来了解它们与血液蛋白结合的紧密程度。这些结果将让我们更好地理解生活规则。考虑到19F核磁共振的上述优点，这也是一种实时研究生化反应和转化的优秀方法。这将被用来方便地测量新的氟化拉曼成像标签和酶探针在整个细胞和细胞液中的生物反应性和稳定性。我们还将使用19F核磁共振，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来促进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易观察到的短暂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和复杂的设备。我们将扩展生物合成工具箱，开发绿色人造酶，这种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，这将彻底改变含氟分子的制备。总体而言，获得专用的冷冻探头，再加上新的分析方法和合成氟化工具，将释放19F核磁共振作为研究动态生物过程的工具尚未开发的潜力。

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.