Harnessing Supercoiling to Regulate DNA Activity

米拉的这项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的各种结构和性质进行了长期、严格的研究，达到了顶峰 以及它与拓扑异构酶的相互作用。在细胞内，DNA是超级卷曲的，通常被限制在小DNA中 可以用足够小的超螺旋DNA小环在实验中重现的环路 生物物理和生化分析的范围。我们开发的方法和广博的知识 到目前为止获得的数据将对我们拟议的DNA拓扑异构酶的研究具有非常重要的作用 针对它们的抗微生物和抗癌药物，工程DNA微环作为基因治疗的应用 载体，以及与人类疾病有关的超螺旋诱导的非规范DNA结构。我们会 首次利用最先进的电子冷冻显微镜和冷冻断层摄影术来确定 拓扑异构酶与生理上相关的DNA底物结合。这一方法将与 使用电泳和荧光技术的综合定量分析和分析 超速离心法研究DNA超螺旋如何强烈影响拓扑异构酶与药物的相互作用。 许多拓扑异构酶，特别是那些重要的药物靶点，优先发挥积极的作用。 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。 DNA也是如此，尽管针对拓扑异构酶的化疗药物的研究在很大程度上忽略了 超螺旋DNA对药物作用的影响。我们计划通过以下方式确定有效药物靶点的新抑制剂 首次筛选与正超螺旋DNA结合的活性拓扑异构酶 50亿种不同化合物。接下来，我们将应用我们的创新工具和令人信服的数据来说明超级卷曲是如何， 曲率和序列决定了DNA构象以设计和构建具有特定的， 理想的形状，适合各种临床应用所需的细胞摄取。现有 纳米粒子，如由金或单糖组成的纳米粒子是惰性的；因此，我们建议使用 DNA微环，作为一体的载体和货物，用于基因治疗，以克服许多障碍 有效的基因传递。最后，我们将使用DNA微环来研究超级线圈如何促进 非B-DNA结构的形成，已知影响DNA复制、修复、转录，但它们的 在体内的频率是有争议的。这项工作具有变革性，因为我们的新DNA微环，先进的成像 工具和量化分析将使我们能够获得前所未有和以前无法获得的洞察力 超螺旋DNA的结构和功能。我们的基础研究将继续挑战 DNA被拓扑异构酶被动作用，但却驱动许多关键细胞的范式 流程。此外，该项目还具有大量与抗拓扑异构酶相关的人类治疗应用。 药物疗效，改进的基因治疗提供，并减轻由非B-DNA形式引起的基因组不稳定。

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.