Selection and sensing applications of DNAzymes selective for paramagnetic metal ions

顺磁性金属离子选择性 DNAzyme 的选择和传感应用

• 批准号: 9368105
• 负责人: Yi Lu
• 金额: $ 28.26万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9368105
• 关键词: Affect; Affinity; Antibiotic Resistance; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Models; Catalytic DNA; Cells; Cleaved cell; Complex; Conserved Sequence; Cytosol; DNA; Dependence; Detection; Development; Electron Spin Resonance Spectroscopy; Elements; Escherichia coli; Fluorescence Resonance Energy Transfer; Goals; Health; Heavy Ions; Homeostasis; Host Defense Mechanism; Human; Immunity; In Vitro; Infection; Ions; Iron; Kinetics; Knowledge; Legal patent; Length; Life; Link; Manganese; Measurement; Metal Ion Binding; Metals; Methods; Modeling; Molecular Conformation; Monitor; Mutagenesis; Neurodegenerative Disorders; Nucleotides; Nutritional; Organism; Outcome; Oxidative Stress; Oxidative Stress Pathway; Pathogenesis; Pathway interactions; Performance; Phagocytes; Process; Public Health; Randomized; Regulation; Respiratory Burst; Role; Sampling; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Staphylococcus aureus; Starvation; System; Technology; Time; Titrations; Validation; X-Ray Crystallography; absorption; base; biophysical analysis; biophysical properties; catalyst; combat; cost; design; field study; fight against; flexibility; fluorophore; functional group; high risk; improved; insight; interest; next generation sequencing; novel; novel strategies; oxidation; pathogen; pathogenic bacteria; periplasm; prevent; ratiometric; sensor; spatiotemporal; stoichiometry; three dimensional structure; tool; uptake

Project summary / abstract The overall goal of this project is to develop and validate a novel class of fluorescent sensors for paramagnetic metal ions (PMIs, e.g., Fe2+, Fe3+, Mn2+ and Mn3+), and to use these sensors to provide deeper insight into the uptake and homeostasis of PMIs in bacteria and the role of PMIs in pathogenesis. PMIs are essential elements for both humans and bacteria; the availability of these metal ions is sharply limited for pathogens, as a part of a host defense mechanism known as “nutritional immunity”; the most well characterized examples being Fe and Mn sequestration during infection. Moreover, Fe and Mn-regulated pathways are closely linked with pathways involved in managing oxidative stress, as occurs in phagocytic respiratory burst. Despite the importance of PMIs in nutritional immunity and oxidative stress pathways, the precise mechanisms dictating nutritional immunity, bacterial uptake of PMIs, and the ability of certain bacterial strains to circumvent metal starvation and thrive are unclear. A major barrier to understanding these complex mechanisms is the lack of spatiotemporal detection of PMIs in their different OSs in living bacterial cells. This proposal seeks to overcome this major barrier by selection and characterization of PMI-specific DNAzymes, and subsequent development and validation of DNAzyme-based turn-on fluorescent sensors selective not only for different PMIs, but also different oxidation states of the same PMI in two model systems (Staphylococcus aureus and Escherichia coli). Specifically, we plan to employ in vitro selection to obtain DNAzymes with high cleavage activity and strong affinity for different PMIs (Fe2+ and Mn2+), while maintaining specificity for the different oxidation states of the same metal ion (Fe2+ vs. Fe3+, and Mn2+ vs. Mn3+). Biochemical studies of these DNAzymes will provide information about conserved sequences, pH and metal ion dependence, and kinetic parameters of the DNAzyme activity. Biophysical characterization using spectroscopic methods (UV-vis and EPR) and x-ray crystallography will elucidate PMI-binding stoichiometry, affinity and selectivity in these DNAzymes. The knowledge acquired will be used to convert these DNAzymes into PMI sensors using the patented catalytic beacon technology. The use of a “caged” and FRET DNAzyme sensor enabling quantitative monitoring of metal ion concentration and speciation in living cells under temporal control will also be explored. Since pathogenic bacteria such as S. aureus and E. coli are a major public health issue, especially due to the spread of antibiotic resistance, our ability to develop turn-on fluorescent sensors for the real time detection of PMIs in cells will overcome a major barrier within the field of nutritional immunity by improving our understanding of the uptake and homeostasis of PMIs in bacteria and the role of PMIs in pathogenesis. Ultimately, knowledge gained from these sensors could provide insights necessary to develop novel strategies to fight against bacterial infection.

项目摘要 /摘要 该项目的总体目标是开发和验证一类新型的荧光传感器 顺磁金属离子（例如PMI，例如Fe2+，Fe3+，Mn2+和Mn3+），并使用这些传感器提供更深的 对细菌中PMI的摄取和稳态的洞察力以及PMI在发病机理中的作用。 PMI是 人类和细菌的基本要素；这些金属离子的可用性受到了极大的限制 病原体，作为一种被称为“营养免疫”的宿主防御机制的一部分；最有特点的 示例是感染过程中FE和MN固相。此外，FE和MN调节的途径紧密 与吞噬呼吸道爆发中发生的涉及氧化应激的途径有关。尽管 PMI在营养免疫和氧化应激途径中的重要性，确切的机制决定了 营养免疫力，细菌吸收PMI和某些细菌菌株绕过金属的能力 饥饿和繁荣不清楚。理解这些复杂机制的主要障碍是缺乏 在活细胞中，PMI在其不同OSS中的时空检测。该提议试图克服 通过选择和表征PMI特异性dnazymes以及随后的发展，这一主要障碍 以及验证基于Dnazyme的荧光传感器，不仅针对不同的PMI，还可以选择 在两个模型系统（金黄色葡萄球菌和大肠杆菌）中，同一PMI的不同氧化态。 具体而言，我们计划采用体外选择以获得具有高裂解活性和强大乳化性的dnazymes 对不同PMI（Fe2+和MN2+）的亲和力，同时保持对不同氧化状态的特异性 相同的金属离子（Fe2+ vs. Fe3+，Mn2+ vs. Mn3+）。这些dnazymes的生化研究将提供 有关配置序列，pH和金属离子依赖性以及Dnazyme的动力学参数的信息 活动。使用光谱法（UV-VIS和EPR）和X射线晶体学的生物物理表征 将阐明这些dnazymes中的PMI结合化学计量，亲和力和选择性。获得的知识将 使用获得专利的催化信标技术将这些dnazymes转换为PMI传感器。使用 “笼”和fret dnazyme传感器，可实现金属离子浓度的定量监测和 还将探索在临时控制下的活细胞规范。 由于金黄色葡萄球菌和大肠杆菌等致病细菌是一个主要的公共卫生问题，尤其是由于 抗生素耐药性的传播，我们开发转交荧光传感器以实时检测的能力 细胞中的PMI将通过改善我们的营养免疫领域中的主要障碍 了解PMI在细菌中的摄取和体内稳态以及PMI在发病机理中的作用。 最终，从这些传感器中获得的知识可以提供开发新型策略所需的见解 与细菌感染作斗争。