Selection and sensing applications of DNAzymes selective for paramagnetic metal ions

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，Fe 2+、Fe 3+、Mn 2+和Mn 3+），并且使用这些传感器来提供更深的 了解细菌中PMI的摄取和稳态以及PMI在发病机制中的作用。编码矩阵指示 人体和细菌的必需元素;这些金属离子的可用性非常有限， 病原体，作为宿主防御机制的一部分，被称为“营养免疫”; 实例是感染期间的Fe和Mn螯合。此外，铁和锰调节途径密切相关， 与参与管理氧化应激的途径相关，如发生在吞噬呼吸爆发中。尽管 PMIs在营养免疫和氧化应激途径中的重要性， 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 饥饿和繁荣是不清楚的。理解这些复杂机制的一个主要障碍是缺乏 在活的细菌细胞中在其不同OS中的PMI的时空检测。该提案旨在克服 这一主要障碍是通过选择和表征PMI特异性DNA酶，以及随后的开发 和基于DNA酶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 在两个模型系统（金黄色葡萄球菌和大肠杆菌）中相同PMI的不同氧化态。 具体而言，我们计划采用体外筛选，以获得具有高切割活性和强切割活性的DNA酶。 对不同PMI（Fe 2+和Mn 2+）的亲和力，同时保持对不同氧化态的PMIs的特异性。 相同的金属离子（Fe 2+与Fe 3+，Mn 2+与Mn 3+）。这些DNA酶的生化研究将提供 关于DNA酶的保守序列、pH和金属离子依赖性以及动力学参数的信息 活动使用光谱学方法（UV-vis和EPR）和X射线晶体学进行生物物理表征 将阐明PMI结合的化学计量，亲和力和选择性在这些DNA酶。获得的知识将 使用专利催化信标技术将这些DNA酶转化为PMI传感器。使用 “笼”和FRET DNAzyme传感器，能够定量监测金属离子浓度， 此外，本课程亦会探讨活细胞在时间控制下的物种形成。 由于致病菌如S. aureus和E.大肠杆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，我们开发用于真实的时间检测的开启荧光传感器的能力 细胞中PMI的增加将通过改善我们的免疫力来克服营养免疫领域内的一个主要障碍。 了解PMI在细菌中的摄取和稳态以及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为开发新策略提供必要的见解 来对抗细菌感染。

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.