Harnessing Supercoiling to Regulate DNA Activity

利用超螺旋调节 DNA 活性

基本信息

批准号：
10205924
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
项目类别：
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/10205924
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/antagonist innovation insight nanoparticle nervous system disorder novel novel therapeutics repaired screening targeted agent three dimensional structure tomography tool uptake

项目摘要

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.

这个 MIRA 提案展示了我对未来五年的研究将如何发展的愿景，以及我们对超螺旋 DNA 的多种结构和特性进行了长期、严格的研究及其与拓扑异构酶的相互作用。在细胞内，DNA 呈超螺旋状，通常被限制成小 DNA 可以用足够小的超螺旋 DNA 微环在实验上重现这些环，以便在广泛的应用中使用一系列生物物理和生化测定。我们开发的方法和广泛的知识迄今为止获得的信息对于我们提出的 DNA 拓扑异构酶、重要作用的研究将是无价的。针对它们的抗菌剂和抗癌剂，工程 DNA 小环作为基因治疗的用途载体，以及超螺旋诱导的与人类疾病有关的非规范 DNA 结构。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进行综合定量测定和分析超速离心来表征 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，特别是那些作为重要药物靶点的拓扑异构酶，优先发挥积极作用超螺旋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研究在很大程度上忽视了超螺旋DNA对药物作用的影响。我们计划通过以下方式识别经过验证的药物靶点的新抑制剂首次针对超螺旋 DNA 文库筛选与正超螺旋 DNA 结合的活性拓扑异构酶 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来了解超螺旋如何，曲率和序列决定 DNA 构象，以设计和构建具有特定、适合各种临床应用所需的细胞摄取的理想形状。现存的纳米颗粒，例如由金或单糖组成的纳米颗粒，是惰性的；因此，我们建议利用 DNA小环作为载体和货物合二为一，用于基因治疗克服许多障碍有效的基因传递。最后，我们将利用 DNA 小环来研究超螺旋如何促进非 B-DNA 结构的形成，已知会影响 DNA 复制、修复、转录，但它们的作用体内频率存在争议。这项工作具有变革性，因为我们新颖的 DNA 小环、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、以前无法获得的见解深入了解超螺旋 DNA 的结构和功能。我们的基础研究将继续挑战 DNA 被动地受到拓扑异构酶的作用，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大量人类治疗应用药物疗效、改善基因治疗递送以及减轻非 B-DNA 形式引起的基因组不稳定性。