A Dynamic Perspective on DNA-binding

小分子和蛋白质与DNA的结合是生物学的基础，高度序列选择性的DNA结合分子作为新的候选药物和操纵基因表达的生物技术工具存在相当大的发展空间。在本提案中，我们重点应用STFC中央激光设备（CLF）开发的尖端时间分辨光谱方法，实时观察B-DNA次要凹槽中小分子与目标序列的结合。由我们的项目合作伙伴MGB生物制药有限公司生产的一种MGB化合物已进入临床前试验阶段，作为对抗医院获得性感染（如艰难梭菌）的新型抗生素，次要凹槽结合（MGB）物种已显示出前景。对这类分子中支撑配体结合的机制有一个全面的分子水平的理解是至关重要的，以便为当前候选分子的改进提供信息，并为癌症治疗等领域的应用设计新的衍生物。然而，尽管进行了大量的研究，关键的悬而未决的问题仍然是关于特定的DNA碱基序列是如何被配体识别的，以及水在促进、介导或抑制配体与DNA结合中的作用。我们知识上的差距是因为目前DNA结合的图像来自x射线衍射、核磁共振或生化分析等实验，至关重要的是，这些实验对DNA结构的快速波动或溶液中溶剂分子驱动的动力学不敏感。这些动力学直接影响结合位点的形状和化学环境，因此必须将它们构建到任何DNA结合模型中。由于CLF和stfc资助的项目访问研究对超快激光技术的资本投资，现在存在使用2D-IR光谱实时和高空间分辨率研究生物分子过程的能力。我们试图通过应用这项技术来记录DNA的第一个“分子电影”：MGB候选药物的配体结合过程，从而确定这项技术可以解决制药领域的关键问题。在此过程中，我们将以前所未有的细节揭示DNA波动和水分子对结合的影响，并证明stfc资助的研究可以率先将学术研究转化为这一领域的影响。这项工作的结果将明显有助于医疗保健MGB物种的设计，我们设想将这些新知识早期整合到药物设计过程中，最终导致下一代候选药物具有更高的功效和选择性，用于其特定的靶标序列。此外，这种能力的展示将为STFC研究和制药部门之间的未来合作提供一个门户，并为利用以前无法获得的来源（如MRC和Wellcome Trust）的未来资助途径提供一个门户。

The binding of small molecules and proteins to DNA is fundamental to biology and considerable scope exists for the development of highly-sequence-selective, DNA-binding molecules as new drug candidates and biotechnological tools to manipulate gene expression. In this proposal we focus on applying cutting-edge time-resolved spectroscopy methods developed at the STFC Central Laser Facility (CLF) to observe, in real time, the binding of small molecules to target sequences in the minor groove of B-DNA. Minor groove binding (MGB) species have shown promise as novel antibiotics for the fight against hospital acquired infections such as Clostridium difficile, with one such MGB compound, produced by our project partner MGB Biopharma Ltd, entering preclinical trials. Obtaining, a comprehensive molecular-level understanding of the mechanisms that underpin ligand binding in this class of molecules is now critical in order to inform refinement of current candidate molecules and the design of new derivatives for applications in areas such as cancer treatment. Despite much study however, key outstanding questions remain regarding the way in which specific DNA base sequences are identified by the ligand and the role of water in promoting, mediating or inhibiting ligand binding to DNA. The gaps in our knowledge arise because the current picture of DNA binding stems from experiments such as X-ray diffraction, NMR or biochemical assays that, crucially, are not sensitive to the rapid fluctuations of the DNA architecture or the solvent molecule-driven dynamics that occur in solution. These dynamics directly influence both the shape and chemical environment of the binding site and it is therefore imperative that they are built into any models of DNA binding.As a result of capital investment in ultrafast laser technology at the CLF and STFC-funded Programme Access research, the capability now exists for studying biomolecular processes in real time and at high spatial resolution using 2D-IR spectroscopy. We seek to establish that this technology can address key issues in the pharmaceutical sector by applying it to record the first 'molecular movie' of the DNA:ligand binding process of an MGB drug candidate. In doing so, we will reveal the influence of DNA fluctuations and water molecules upon binding in unprecedented detail and demonstrate that STFC-funded research can take a lead in transferring academic research to impact in this arena. The results of this work will demonstrably contribute to the design of MGB species for healthcare and we envisage integrating this new knowledge early into the drug design process, ultimately leading to next generation drug candidates with improved efficacy and selectivity for their particular target sequences. In addition, this demonstration of capability will provide a gateway both for future engagement between STFC research and the pharmaceutical sector and for exploitation of future funding routes from previously inaccessible sources such as MRC and the Wellcome Trust.