Selection, Characterization & Application of Paramagnetic Metal-specific DNAzymes

描述(申请人提供)：脱氧核酶或脱氧核酶是具有酶活性的DNA分子。自1994年发现以来，DNAzyme已被证明是一种金属酶，可以转化为金属离子传感器。从科学上讲，虽然金属离子在蛋白质中的作用已经积累了大量的知识，但对核酸的了解要少得多。在技术上，虽然抗磁性金属离子传感器的设计已经取得了巨大的进展，但设计顺磁性金属离子传感器，特别是同一金属离子的不同氧化态仍然具有挑战性。该项目寻求通过推进DNAzyme中金属结合位点的科学知识来填补这两个空白，并扩大其作为顺磁性金属离子传感器的技术应用，以改善环境健康。具体地说，我们首先计划通过体外筛选获得对磷酸二酯转移具有高活性并且对不同顺磁性金属离子(Co2+、Cu2+或Fe2+)或同一金属离子(Fe2+与Fe3+)的不同氧化状态具有很强亲和力的DNAzyme。对选定的DNAzyme的生化研究将提供关于保守序列、催化参数以及酶活性的pH和金属离子依赖性的信息。利用UV-Vis、EPR、MCD、XAS、FRET和X射线结晶学进行生物物理表征，将阐明这些DNAzyme中金属结合部位的亲和力、化学计量比、几何构型和配体供体集，以及反应中间体和反应机理。在这个过程中获得的知识将被用来利用专利催化信标技术将这些DNAzyme转化为灵敏和选择性的金属传感器。如果这个项目的目标得以实现，我们将进一步了解金属离子在每个被研究的DNA酶中的作用，以及不同的结构特征如何影响酶的活性。这将使我们对DNAzyme中金属离子的理解水平更接近于蛋白质中的金属离子。它还将提供一个独特的机会来比较和对比蛋白质和DNA中相同金属离子(如Cu2+或Fe2+)的结构和功能特性，这将是令人着迷的，因为蛋白质和DNAzyme使用非常不同的构建块。此外，专利催化信标方法对多种顺磁性金属离子(包括同一金属离子的不同氧化状态)的普遍适用性的展示将推动环境健康领域的发展，使环境监测、发育生物学、临床毒理学、废水处理和工业监测中的金属离子能够进行现场、实时检测。最后，从基础配位化学的研究中获得的见解将为基于有机分子、聚合物或多肽的其他类型的金属传感器的合理设计提供参考。它还将对传感器设计以外的研究领域产生重要影响，如过渡金属核酸酶和药物制剂的设计。与公众健康相关：顺磁性金属离子，如钴、铜和铁，在低浓度时对人体健康有益，但在高浓度时有毒。为这些金属离子开发便携式荧光DNAzyme传感器将推动环境健康领域的发展，使环境监测、发育生物学、临床毒理学、废水处理和工业监测中的金属离子能够进行现场、实时检测。这项研究获得的见解将有助于其他类型金属传感器的合理设计，并可能对其他研究领域产生影响，如过渡金属核酸酶和药物制剂的设计。

DESCRIPTION (provided by applicant): DNAzymes, or deoxyribozymes are DNA molecules with enzymatic activities. Since its discovery in 1994, DNAzymes have been shown to be metalloenzymes and can be converted into metal ion sensors. Scientifically, whereas a great deal of knowledge has been accumulated in the roles of metal ions in proteins, much less is known in nucleic acids. Technologically, while enormous progress has been made in the designing sensors for diamagnetic metal ions, designing sensors for paramagnetic metal ions, particularly different oxidation states of the same metal ions remains challenging. The project seeks to fill both gaps by advancing scientific knowledge of metal-binding sites in DNAzymes, and expanding their technological applications as paramagnetic metal ion sensors that will be used to improve environmental health. Specifically we first plan to employ in vitro selection to obtain DNAzymes with high activity toward phosphodiester transfer and with strong affinity for different paramagnetic metal ions (Co2+, Cu2+ or Fe2+), or different oxidation states of the same metal ion (Fe2+ vs. Fe3+). Biochemical studies of the selected DNAzymes will provide information about conserved sequence, catalytic parameters, and pH and metal ion dependence of the enzyme activity. Biophysical characterization using UV-vis, EPR, MCD, XAS, FRET, and X-ray crystallography will elucidate affinity, stoichiometry, geometry,and ligand donor sets of the metal-binding sites in these DNAzymes, as well as reaction intermediates and mechanism. The knowledge acquired in this process will be used to convert these DNAzymes into sensitive and selective metal sensors using a patented catalytic beacon technology. If the aims of this project are achieved, we will advance scientific knowledge of the roles of metal ions in each DNAzyme investigated and how different structural features influence the enzyme activity. It will bring our level of understanding of metal ions in DNAzymes closer to that in proteins. It will also allow a unique opportunity to compare and contrast structural and functional properties of the same metal ions, such as Cu2+ or Fe2+, in proteins and in DNA, which will be fascinating because proteins and DNAzymes use very different building blocks. Furthermore, the demonstration of general applicability of the patented catalytic beacon method to sense a wide variety of paramagnetic metal ions (including different oxidation states of the same metal ions) will drive the field of environmental health, allowing on-site, real-time detection of metal ions in environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial monitoring. Finally, the insight gained from the study on the basic coordination chemistry will shed light on rational design of other types of metal sensors based on organic molecules, polymers or peptides. It will also have important impact on research areas beyond sensor design, such as the design of transition metal-based nucleases and pharmaceutical agents. PUBLIC HEALTH RELEVANCE: Paramagnetic metal ions such as cobalt, copper and iron are beneficial to human health when low in concentration, but are toxic when high in concentration. Developing portable fluorescent DNAzyme sensors for these metal ions will advance the field of environmental health, allowing on-site, real-time detection of metal ions in environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial monitoring. Insights gained from the study will shed light on rational design of other types of metal sensors and could impact on other research areas such as the design of transition metal-based nucleases and pharmaceutical agents.