Harnessing Supercoiling to Regulate DNA Activity

利用超螺旋调节 DNA 活性

• 批准号: 10482361
• 负责人: LYNN ZECHIEDRICH
• 金额: $ 40万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10482361
• 关键词: Address; Affect; Antibiotics; Antineoplastic Agents; Arts; Binding; Biochemical; Biological Assay; Biophysics; Cell physiology; Cells; Chromatin Loop; Coupled; Cryoelectron Microscopy; DNA; DNA Structure; DNA Topoisomerases; DNA biosynthesis; Data; Drug Interactions; Drug Targeting; Engineering; Enzymes; Fluorescence; Frequencies; Gene Delivery; Gene Transduction Agent; Genetic Transcription; Genomic Instability; Gold; Human; Imaging Device; Infection; Knowledge; Libraries; Malignant Neoplasms; Methods; Molecular Conformation; Monosaccharides; Pharmaceutical Preparations; Physiological; Property; Research; Sculpture; Shapes; Structure; Superhelical DNA; Techniques; Textbooks; Therapeutic; Time; Topoisomerase; Vision; Work; analytical ultracentrifugation; antimicrobial drug; clinical application; design and construction; drug action; drug efficacy; experimental study; fundamental research; gene therapy; human disease; improved; in vivo; inhibitor; innovation; insight; nanoparticle; nervous system disorder; novel; novel therapeutics; repaired; screening; targeted agent; three dimensional structure; tomography; tool; uptake

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.

这项Mira提议介绍了我对我的研究在未来五年中如何发展的愿景， 从我们长期，严格的研究超冰DNA的多种结构和特性中达到高潮 及其与拓扑异构酶的相互作用。在细胞内，DNA被超冰，通常被约束到小的DNA中 可以在实验中用超螺旋的DNA小圆圈在实验中概括的循环，足以在宽阔的地方使用 生物物理和生化测定范围。我们开发的方法和广泛的知识 到目前为止获得的获取对于我们提出的对DNA拓扑酶的研究将是无价的 靶向它们的抗菌剂和抗癌剂，工程DNA微圆作为基因疗法的效用 与人类疾病有关的载体和超螺旋诱导的非规范DNA结构。我们将 首先利用最先进的电子冷冻微观镜和冷冻术来确定的3-D结构 拓扑异构酶与生理相关的DNA底物结合。这种方法将与 使用电泳和荧光技术以及分析的综合定量测定 超速离心以表征DNA超螺旋如何强烈影响拓扑异构酶 - 毒物相互作用。 许多拓扑异构酶，尤其是那些重要的药物靶标的拓扑异构酶，优先采取积极作用 超螺旋DNA。因此，相应的抗昆虫异构酶药物与阳性超冰相互作用 DNA也是DNA，尽管针对拓扑异构酶的化学治疗剂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 筛选首次筛选活跃的拓扑异构酶，绑定到超过超螺旋的DNA与一个库的库 50亿种不同的化合物。接下来，我们将应用我们的创新工具和令人信服的数据，以了解超统一的方式， 曲率和序列决定DNA构象以设计和构建具有特定的DNA纳米颗粒 所需的形状非常适合各种临床应用中所需的细胞摄取。现存的 纳米颗粒，例如由黄金或单糖组成的纳米颗粒是惰性的。因此，我们建议利用 DNA微圆，作为车辆和货物，用于基因治疗，以克服许多障碍 有效的基因输送。最后，我们将采用DNA微圆来研究超螺旋如何促进 已知会影响DNA复制，修复，转录的非B-DNA结构的形成，但 体内频率是有争议的。这项工作是变革性的，就像我们的新型DNA微圆，高级成像 工具和定量分析将使我们能够实现前所未有且以前无法实现的见解 进入超螺旋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的范例，而是驱动许多关键细胞 过程。此外，该项目具有与抗昆虫异构酶有关的大量人类治疗应用 药物疗效，改善基因治疗的递送以及由非B-DNA形式引起的基因组不稳定性。