Selection, Characterization & Application of Paramagnetic Metal-specific DNAzymes

选择、表征

基本信息

批准号：
8272648
负责人：
Yi Lu
金额：
$ 35.01万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-07-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8272648
关键词：
Affinity Area Binding Biochemical Catalytic DNA Chemistry Cobalt Collection Conserved Sequence Copper DNA DNA Microarray Chip Dependence Detection Development Developmental Biology Electrochemistry Electron Spin Resonance Spectroscopy Electronics Environmental Health Environmental Monitoring Enzymes Family Fluorescence Resonance Energy Transfer Goals Health Human In Vitro Individual Ions Iron Kinetics Knowledge Label Legal patent Length Ligands Light Mass Spectrum Analysis Metal Binding Site Metals Methods Monitor Mutagenesis Nucleic Acids Nucleotides Oxidation-Reduction Peptides Pharmacologic Substance Polymers Process Property Proteins Reaction Research Roentgen Rays Role Science Scientific Advances and Accomplishments Sequence Determination Site Site-Directed Mutagenesis Solutions Spectrum Analysis Techniques Technology Testing Time Transition Elements X-Ray Crystallography absorption base catalyst circular magnetic dichroism clinical toxicology design enzyme activity fascinate fluorophore improved in vitro Assay insight member metalloenzyme nuclease oxidation phosphodiester sensor single molecule stoichiometry

项目摘要

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.

描述（由申请人提供）：dnazymes或脱氧核酶是具有酶促活性的DNA分子。自1994年发现以来，dnazymes已被证明是金属酶，可以转化为金属离子传感器。从科学上讲，尽管金属离子在蛋白质中的作用中积累了大量知识，但在核酸中却少得多。从技术上讲，虽然在设计传感器的磁管金属离子的设计传感器中取得了巨大进展，但为顺磁金属离子设计传感器，尤其是同一金属离子的氧化态，仍然具有挑战性。该项目旨在通过促进dnazymes中金属结合位点的科学知识来填补这两个空白，并扩大其技术应用，作为顺磁金属离子传感器，这些传感器将用于改善环境健康。具体而言，我们首先计划采用体外选择来获得具有较高活性的dnazymes，其对磷酸二酯转移的活性很高，并且对不同的顺磁金属离子（CO2+，Cu2+或Fe2+）或同一金属离子的不同氧化态具有很强的亲和力（Fe2+vs. Fe3+）。所选dnazymes的生化研究将提供有关酶活性的保守序列，催化参数以及pH和金属离子依赖性的信息。使用UV-VIS，EPR，MCD，XAS，FRET和X射线晶体学的生物物理表征将阐明这些DNAZEMES中金属结合位点的亲和力，石化，几何和配体供体集，以及反应中间体和机械机制和机械机制。在此过程中获得的知识将使用专利的催化信标技术将这些dnazymes转换为敏感和选择性的金属传感器。如果实现了该项目的目的，我们将提高有关金属离子在所研究的每个dnazyme中的作用以及不同结构特征如何影响酶活性的科学知识。它将使我们对蛋白质中脂肪素的金属离子的理解水平。它还将允许在蛋白质和DNA中比较和对比相同金属离子（例如Cu2+或Fe2+）的结构和功能性能的独特机会，因为蛋白质和dnazymes使用截然不同的构造块，这将引人入胜。此外，专利的催化信标方法的一般适用性表明，感知多种顺磁金属离子（包括同一金属离子的不同氧化态）将推动环境健康领域，从而可以在环境监测，发育生物学，临床毒性，临床监测和工业中对环境监测的现场，实时对金属离子进行实时检测。最后，从基本协调化学研究中获得的洞察力将阐明基于有机分子，聚合物或肽的其他类型的金属传感器的合理设计。它还将对传感器设计以外的研究领域产生重要影响，例如过渡金属的核酸酶和药物的设计。公共卫生相关性：顺磁金属离子（例如钴，铜和铁）在浓度较低时对人类健康有益，但浓度较高时具有毒性。为这些金属离子开发便携式荧光DNAZYME传感器将促进环境健康领域，从而可以在环境监测，发育生物学，临床毒理学，废水治疗和工业监测中对现场，实时检测金属离子。从研究中获得的洞察力将阐明其他类型的金属传感器的合理设计，并可能影响其他研究领域，例如过渡金属的核酸酶和药物的设计。