Harnessing Supercoiling to Regulate DNA Activity

这个MIRA提案提出了我对未来五年我的研究将如何发展的愿景， 我们对超螺旋DNA的不同结构和性质进行了长期、严格的研究， 及其与拓扑异构酶的相互作用。在细胞内，DNA是超螺旋的，通常被限制成小DNA 环，可以实验重演超螺旋DNA微环小到足以用于广泛的 一系列生物物理和生物化学测定。我们开发的方法和广泛的知识 获得迄今将是非常宝贵的，我们提出的研究DNA拓扑异构酶，行动的重要 靶向它们的抗微生物剂和抗癌剂，工程化DNA微环作为基因治疗的效用 载体和超螺旋诱导的非规范DNA结构，这些结构与人类疾病有关。我们将 首先利用最先进的电子冷冻显微镜和冷冻断层扫描来确定 拓扑异构酶结合到生理相关的DNA底物。这种方法将与 使用电泳和荧光技术的综合定量分析和分析 超离心来表征DNA超螺旋如何如此强烈地影响拓扑异构酶-药物相互作用。 许多拓扑异构酶，特别是那些重要的药物靶标，优先作用于 超螺旋DNA因此，相应的抗拓扑异构酶药物与正超螺旋相互作用， 尽管靶向拓扑异构酶的化疗药物的研究在很大程度上忽视了DNA， 超螺旋DNA对药物作用影响我们计划通过以下方法来确定经验证的药物靶点的新抑制剂： 第一次筛选与正超螺旋DNA结合的活性拓扑异构酶， 50亿多种化合物。接下来，我们将应用我们的创新工具和令人信服的数据， 曲率和序列决定了DNA构象， 这些形状对于各种临床应用中所需的细胞摄取是理想的。现有 纳米颗粒，如由金或单糖组成的那些，是惰性的;因此，我们建议利用 DNA微环，作为载体和货物在一个，基因治疗，以克服许多障碍， 有效的基因传递。最后，我们将利用DNA微环来研究超螺旋如何促进DNA的超螺旋化。 非B-DNA结构的形成，这是已知的影响DNA复制，修复，转录，但它们的 体内频率是有争议的。这项工作是变革性的，因为我们的新型DNA微环，先进的成像技术， 工具和定量分析将使我们能够获得前所未有的和以前无法实现的见解 超螺旋DNA的结构和功能我们的基础研究将继续挑战 DNA是被动地作用于拓扑异构酶，而是驱动许多关键的细胞 流程.此外，该项目具有与抗拓扑异构酶相关的实质性人类治疗应用 药物功效、改进的基因治疗递送和减轻由非B-DNA形式引起的基因组不稳定性。

This MIRA proposal presents my vision for how my research will evolve over the next five years and culminates from our long-term, rigorous studies of the diverse structures and properties of supercoiled DNA and its interaction with topoisomerases. Within cells, DNA is supercoiled and often constrained into small DNA loops that can be experimentally recapitulated with supercoiled DNA minicircles small enough for use in a wide range of biophysical and biochemical assays. The methods we have developed and extensive knowledge acquired thus far will be invaluable for our proposed studies of DNA topoisomerases, actions of important antimicrobial and anticancer agents that target them, the utility of engineered DNA minicircles as gene therapy vectors, and supercoiling-induced noncanonical DNA structures that are implicated in human disease. We will first utilize state-of-the-art electron cryo-microscopy and cryo-tomography to determine the 3-D structure of topoisomerases bound to physiologically relevant DNA substrates. This approach will be coupled with comprehensive quantitative assays using electrophoretic and fluorescence techniques and analytical ultracentrifugation to characterize how DNA supercoiling so strongly affects topoisomerase-drug interactions. Many topoisomerases, particularly those that are important drug targets, preferentially act on positively supercoiled DNA. Consequently, corresponding anti-topoisomerase drugs interact with positively supercoiled DNA as well, although research of chemotherapeutics that target topoisomerases has largely disregarded the effect of supercoiled DNA on drug action. We plan to identify new inhibitors of validated drug targets by screening, for the first time, active topoisomerase bound to positively supercoiled DNA against a library of over 5 billion diverse compounds. We will next apply our innovative tools and compelling data of how supercoiling, curvature, and sequence dictate DNA conformation to design and construct DNA nanoparticles with specific, desired shapes that are ideal for cellular uptake needed in a variety of clinical applications. Existing nanoparticles, such as those composed of gold or monosaccharides, are inert; therefore, we propose utilizing DNA minicircles, as both the vehicle and cargo in one, for gene therapy to overcome many of the barriers to effective gene delivery. Finally, we will employ DNA minicircles to investigate how supercoiling promotes the formation of non-B-DNA structures, which are known to impact DNA replication, repair, transcription, yet their in vivo frequency is controversial. This work is transformative, as our novel DNA minicircles, advanced imaging tools, and quantitative analyses will enable us to achieve unprecedented and previously unattainable insights into the structure and function of supercoiled DNA. Our fundamental research will continue to challenge the paradigm that DNA is passively acted upon by topoisomerases but instead drives numerous critical cellular processes. Moreover, this project has substantial human therapeutic applications related to anti-topoisomerase drug efficacy, improved gene therapy delivery, and mitigating genomic instability caused by non-B-DNA forms.