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: 9908095
• 负责人: Yi Lu
• 金额: $ 25.74万
• 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 2021-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908095
• 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; 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 Immunity; 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; 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.

项目概要/摘要 该项目的总体目标是开发和验证一类新型荧光传感器 顺磁性金属离子（PMI，例如 Fe2+、Fe3+、Mn2+ 和 Mn3+），并使用这些传感器提供更深层次的检测 深入了解细菌中 PMI 的摄取和稳态以及 PMI 在发病机制中的作用。 人类和细菌的必需元素；这些金属离子的可用性非常有限 病原体，作为被称为“营养免疫”的宿主防御机制的一部分； 例如感染期间铁和锰的隔离。此外，铁和锰调节的途径密切相关。 尽管发生在吞噬细胞呼吸爆发中，但与管理氧化应激的途径有关。 PMI 在营养免疫和氧化应激途径中的重要性，其精确机制决定了 营养免疫、细菌对 PMI 的摄取以及某些细菌菌株规避金属的能力 饥饿和繁荣尚不清楚，理解这些复杂机制的一个主要障碍是缺乏。 该提案旨在克服活细菌细胞中不同操作系统中 PMI 的时空检测问题。 通过选择和表征 PMI 特异性 DNAzyme 以及后续开发来克服这一主要障碍 基于 DNAzyme 的开启荧光传感器的验证不仅对不同的 PMI 具有选择性，而且对 相同 PMI 在两个模型系统（金黄色葡萄球菌和大肠杆菌）中的不同氧化态。 具体来说，我们计划采用体外选择来获得具有高裂解活性和强的DNAzymes。 对不同 PMI（Fe2+ 和 Mn2+）的亲和力，同时保持对不同氧化态的特异性 相同的金属离子（Fe2+ 与 Fe3+，以及 Mn2+ 与 Mn3+）将提供这些 DNAzyme 的生化研究。 有关 DNAzyme 的保守序列、pH 和金属离子依赖性以及动力学参数的信息 使用光谱方法（UV-vis 和 EPR）和 X 射线晶体学进行生物物理表征。 将阐明这些 DNAzyme 中 PMI 结合的化学计量、亲和力和选择性。 使用专利催化信标技术将这些 DNAzyme 转化为 PMI 传感器。 “笼式”和 FRET DNAzyme 传感器能够定量监测金属离子浓度和 还将探讨时间控制下活细胞的物种形成。 由于金黄色葡萄球菌和大肠杆菌等致病菌是一个重大的公共卫生问题，特别是由于 抗生素耐药性的蔓延，我们开发用于实时检测的开启荧光传感器的能力 细胞中的 PMI 将通过改善我们的免疫系统来克服营养免疫领域的主要障碍 了解细菌中 PMI 的摄取和稳态以及 PMI 在发病机制中的作用。 最终，从这些传感器获得的知识可以提供开发新策略所需的见解 以对抗细菌感染。