Unravelling the invisible complexities of the genome

罗莎琳德·富兰克林（Rosalind Franklin）建立DNA原子结构的开创性工作为我们对“生命分子”的理解奠定了基础，然而在细胞中，DNA是纠缠和扭曲的，采用复杂的拓扑结构，并且经常在超螺旋压力下维持。盘绕、扭曲和打结对复杂基因组DNA的影响影响其功能及其与分子机制的相互作用。然而，这种分子的复杂性和灵活性意味着，缠绕和扭曲的DNA的结构和相互作用仍然很不清楚。至关重要的是，我们要提高这种理解，因为构成基因组大部分的复杂DNA结构对我们的健康有巨大的影响：在衰老、癌症和对抗传染病方面。AFM在亚纳米分辨率结构工具中的独特之处在于，它能够在生理温度下对液体中的单个分子进行成像，而生物分子在生理温度下是活跃的（并且可以自由地探索它们的天然构象空间），尽管它们被束缚在表面上。AFM使用一个锋利的探针来“感觉”被吸附在平坦基底上的分子表面，在液体中具有纳米级的精度。我的高分辨率原子力显微镜（AFM）方法在不需要标记或平均的情况下提供DNA结构、功能和动力学的定量信息方面是独一无二的，我的工作证明了DNA沿着单个分子的双螺旋结构的变化。尽管AFM具有在流体中以高分辨率观察单个分子的独特能力，但由于技术的复杂性以及对产生的强大数据的有限分析，其广泛采用受到了限制。传统上，大多数AFM分析都是手工进行的，依靠训练有素且经验丰富的研究人员。再加上数据采集高度依赖于操作人员的专业知识，这意味着AFM还没有被采用为解决目前其他结构生物学工具无法解决的问题的工具，这些工具在这种长度范围内运行。我将率先使用高分辨率AFM和自动化分析来克服这些限制，并揭示DNA结构和构象对DNA-蛋白质相互作用的影响。为了实现这一目标，我将与工业界合作，将最先进的原子力显微镜的发展与新的自动化分析工具结合起来，利用多种机器学习方法，促进对DNA底物的拓扑、结构和构象的跟踪和量化。我将与AFM社区合作，确保这些工具可供各级研究人员使用，提高他们分析的可重复性，并降低这种成像方法的活化能，目前这是一个相当大的进入障碍。使用这些工具，我将确定DNA的结构异质性如何影响其与关键抗生素和抗癌靶点的相互作用：拓扑异构酶；基因编辑工具CRISPR-Cas9；和g -四联体，这两种DNA结构可能成为新的抗癌靶点。该计划侧重于具有转化潜力的系统，使我能够影响药物开发。这个雄心勃勃的计划以我在高分辨率原子力显微镜方面的专业知识为基础，并得到一个多元化的跨学科团队的支持，其中包括一个原子力显微镜制造商，以及互补的单分子生物物理技术、机器学习、分子动力学模拟和生物化学方面的专家。我们将共同揭示基因组中拓扑压力对其整个构象景观相互作用的隐藏影响。这些知识可以通过帮助合理设计新的治疗方法来对抗疾病。

Rosalind Franklin's pioneering work to establish the atomic structure of DNA has underpinned much of our understanding of the 'molecule of life', however in the cell, DNA is tangled and twisted, adopts complex topologies and is frequently maintained under superhelical stress. The effect of coiling, twisting and knotting on complex genomic DNA affects its function and how it interacts with molecular machinery. However the complexity and flexibility of this molecule means that much about the structure and interactions of tangled and twisted DNA remains poorly defined. It is critical that we improve this understanding as complex DNA structures, which make up the majority of the genome, have a huge impact on our health: in aging, cancer and fighting infectious disease. The defining feature of AFM, unique among other structural tools operating at sub-nanometre resolution, is its capacity for imaging single molecules in liquid at physiological temperatures, where biomolecules are active (and free to explore their native conformational space), albeit tethered to a surface. AFM uses a sharp probe to 'feel' the surface of molecules adsorbed on a flat substrate, with nanometre precision in liquid. My high-resolution atomic force microscopy (AFM) methods are unique in their ability to provide quantitative information on DNA structure, function and kinetics without labelling or averaging, demonstrated by my work showing variation in the double-helical structure of DNA along a single molecule. Despite its unique capabilities for observing individual molecules at high resolution in fluid, the widespread adoption of AFM has been limited by the complexity of the technique, and the limited analysis of the powerful data produced. Traditionally, the majority of AFM analysis has been carried out by hand, relying on a highly trained and experienced researcher. When coupled with data acquisition that is highly dependent on the expertise of the operator, this has meant that AFM has not been adopted as the tool that can solve problems currently inaccessible to other tools of structural biology, which operate at this length scale. I will pioneer the use of high-resolution AFM and automated analysis to overcome these limitations and uncover the effect of DNA structure and conformation on DNA-protein interactions. To achieve this I will work in collaboration with industry to combine state-of-the-art atomic force microscopy developments, with new automated analysis tools that facilitate tracing and quantification of the topology, structure and conformation of DNA substrates, using multiple machine learning approaches. I will work with the AFM community to ensure that these tools are available to researchers at all levels, improving the reproducibility of their analysis, and lowering the activation energy for this method of imaging, currently a considerable barrier to entry. Using these tools I will determine how the structural heterogeneity of DNA impacts its interactions with key antibiotic and anti-cancer targets: topoisomerases; the gene editing tool CRISPR-Cas9; and G-quadruplexes, alternative DNA structures with potential as new anti-cancer targets. This programme is focussed on systems with translational potential, to enable me to impact pharmaceutical development. This ambitious programme is underpinned by my expertise in high-resolution AFM and supported by a diverse interdisciplinary team including an AFM manufacturer, and experts in complementary single-molecule biophysics techniques, machine learning, molecular dynamics simulations and biochemistry. Together we will uncover the hidden effect of topological stress in the genome on interactions across its entire conformational landscape. This knowledge can be harnessed to combat disease by aiding in the rational design of novel therapeutics.