Selection, Characterization & Application of Paramagnetic Metal-specific DNAzymes
选择、表征
基本信息
- 批准号:8026609
- 负责人:
- 金额:$ 3.24万
- 依托单位:
- 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 2013-04-30
- 项目状态:已结题
- 来源:
- 关键词:AffinityAreaBindingBiochemicalCatalytic DNAChemistryCobaltCollectionConserved SequenceCopperDNADNA Microarray ChipDependenceDetectionDevelopmentDevelopmental BiologyElectrochemistryElectron Spin Resonance SpectroscopyElectronicsEnvironmental HealthEnvironmental MonitoringEnzymesFamilyFluorescence Resonance Energy TransferGoalsHealthHumanIn VitroIndividualIonsIronKineticsKnowledgeLabelLegal patentLengthLigandsLightMass Spectrum AnalysisMetal Binding SiteMetalsMethodsMonitorMutagenesisNucleic AcidsNucleotidesOxidation-ReductionPeptidesPharmacologic SubstancePolymersProcessPropertyProteinsReactionResearchRoentgen RaysRoleScienceScientific Advances and AccomplishmentsSequence DeterminationSiteSite-Directed MutagenesisSolutionsSpectrum AnalysisTechniquesTechnologyTestingTimeTransition ElementsX-Ray Crystallographyabsorptionbasecatalystcircular magnetic dichroismclinical toxicologydesignenzyme activityfascinatefluorophoreimprovedin vitro Assayinsightmembermetalloenzymenucleaseoxidationphosphodiesterpublic health relevancesensorsingle moleculestoichiometry
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):DNA酶或脱氧核酶是具有酶活性的DNA分子。自1994年发现以来,DNA酶已被证明是金属酶,并可转化为金属离子传感器。科学家们对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,虽然在设计用于抗磁性金属离子的传感器方面已经取得了巨大的进步,但是设计用于顺磁性金属离子的传感器,特别是用于相同金属离子的不同氧化态的传感器仍然具有挑战性。该项目旨在通过推进DNA酶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。具体而言,我们首先计划采用体外选择来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(Co 2+、Cu 2+或Fe 2+)或相同金属离子的不同氧化态(Fe 2 + vs. Fe 3+)具有强亲和力的DNA酶。所选择的DNA酶的生物化学研究将提供有关保守序列,催化参数,pH值和金属离子依赖的酶活性的信息。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DNA酶中的金属结合位点的亲和力,化学计量,几何形状和配体供体集,以及反应中间体和机制。在这一过程中获得的知识将用于使用专利催化信标技术将这些DNA酶转化为敏感和选择性的金属传感器。如果该项目的目标得以实现,我们将推进对金属离子在所研究的每种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。这将使我们对DNA酶中金属离子的理解水平更接近于蛋白质中的水平。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Cu 2+或Fe 2+)的结构和功能特性,这将是非常有趣的,因为蛋白质和DNA酶使用非常不同的构建块。此外,证明专利催化信标方法的普遍适用性,以感测各种顺磁性金属离子(包括相同金属离子的不同氧化态)将推动环境健康领域,允许在环境监测,发育生物学,临床毒理学,废水处理和工业监测中现场实时检测金属离子。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,聚合物或肽的其他类型的金属传感器的合理设计提供启发。它还将对传感器设计以外的研究领域产生重要影响,例如基于过渡金属的核酸酶和药物制剂的设计。公共卫生关系:钴、铜、铁等顺磁性金属离子在低浓度时对人体健康有益,但在高浓度时有毒。开发针对这些金属离子的便携式荧光DNA酶传感器将推动环境健康领域的发展,允许在环境监测、发育生物学、临床毒理学、废水处理和工业监测中现场实时检测金属离子。从这项研究中获得的见解将有助于其他类型金属传感器的合理设计,并可能影响其他研究领域,如过渡金属核酸酶和药物制剂的设计。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
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Yi Lu其他文献
Yi Lu的其他文献
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{{ truncateString('Yi Lu', 18)}}的其他基金
Design and Selection of Novel Metalloenzymes for Biocatalysis, Bioimaging, and Genetic Engineering
用于生物催化、生物成像和基因工程的新型金属酶的设计和选择
- 批准号:
10415131 - 财政年份:2021
- 资助金额:
$ 3.24万 - 项目类别:
Design and Selection of Novel Metalloenzymes for Biocatalysis, Bioimaging, and Genetic Engineering
用于生物催化、生物成像和基因工程的新型金属酶的设计和选择
- 批准号:
10206576 - 财政年份:2021
- 资助金额:
$ 3.24万 - 项目类别:
Design and Selection of Novel Metalloenzymes for Biocatalysis, Bioimaging, and Genetic Engineering
用于生物催化、生物成像和基因工程的新型金属酶的设计和选择
- 批准号:
10673016 - 财政年份:2021
- 资助金额:
$ 3.24万 - 项目类别:
Design and Selection of Novel Metalloenzymes for Biocatalysis, Bioimaging, and Genetic Engineering
用于生物催化、生物成像和基因工程的新型金属酶的设计和选择
- 批准号:
10476760 - 财政年份:2021
- 资助金额:
$ 3.24万 - 项目类别:
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 - 财政年份:2017
- 资助金额:
$ 3.24万 - 项目类别:
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:
10523906 - 财政年份:2017
- 资助金额:
$ 3.24万 - 项目类别:
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:
9368105 - 财政年份:2017
- 资助金额:
$ 3.24万 - 项目类别:
Novel DNAzyme sensors for lithium and sodium to understand cellular and molecular mechanisms of lithium treatment of bipolar disorder
新型锂和钠 DNAzyme 传感器可了解锂治疗双相情感障碍的细胞和分子机制
- 批准号:
9169356 - 财政年份:2016
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$ 3.24万 - 项目类别:
Novel DNAzyme sensors for lithium and sodium to understand cellular and molecular mechanisms of lithium treatment of bipolar disorder
新型锂和钠 DNAzyme 传感器可了解锂治疗双相情感障碍的细胞和分子机制
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9306205 - 财政年份:2016
- 资助金额:
$ 3.24万 - 项目类别:
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