Selection and sensing applications of DNAzymes selective for paramagnetic metal ions

项目摘要/摘要 这个项目的总体目标是开发和验证一种新型的荧光传感器，用于 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 深入了解细菌对PMIs的摄取和动态平衡，以及PMIs在发病机制中的作用。PMI是 对人类和细菌都是必需的元素；这些金属离子的可获得性对于 病原体，作为被称为“营养免疫”的宿主防御机制的一部分；最有特点的 例如，在感染过程中铁和锰被隔离。此外，铁和锰调节的途径是密切的 与管理氧化应激的途径有关，如吞噬细胞呼吸爆发。尽管 PMIs在营养免疫和氧化应激途径中的重要性，其确切的机制决定了 营养免疫、细菌对PMI的摄取以及某些细菌株绕过金属的能力 饥饿和繁荣还不清楚。理解这些复杂机制的一个主要障碍是缺乏 活细菌细胞中不同OSS中PMIs的时空检测。这项提议旨在克服 这一主要障碍是通过选择和鉴定PMI特异性DNAzyme以及随后的开发来实现的 基于DNAzyme的开启荧光传感器的有效性不仅对不同的PMI具有选择性，而且 同一PMI在两个模型体系(金黄色葡萄球菌和大肠杆菌)中的不同氧化状态。 具体地说，我们计划通过体外选择获得具有高切割活性和强切割活性的dna zyme。 对不同的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与Fe3，以及Mn2与Mn3)。对这些脱氧核酶的生化研究将提供 关于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 活动。利用光谱学方法(UV-Vis和EPR)和X射线结晶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。所获得的知识将 利用专利催化信标技术将这些DNAzyme转化为PMI传感器。它的用途 笼式和FRET DNAzyme传感器能够定量监测金属离子浓度和 还将探索在时间控制下的活细胞中的物种形成。 由于金黄色葡萄球菌和大肠杆菌等致病菌是一个主要的公共卫生问题，特别是由于 抗生素耐药性的传播，我们有能力开发开启的荧光传感器进行实时检测 将克服营养免疫领域的一个主要障碍，通过改善我们的 了解细菌对PMIs的摄取和动态平衡，以及PMIs在发病机制中的作用。 最终，从这些传感器获得的知识可以为开发新策略提供必要的见解。 与细菌感染作斗争。

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.