Spectroscopic and Mechanistic Characterization of Novel DNAzymes Selective for Redox-active Metal Ions

选择性氧化还原活性金属离子的新型 DNAzyme 的光谱和机理表征

• 批准号: 10538382
• 负责人: Casey Michael Van Stappen
• 金额: $ 7.23万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-05 至 2025-09-04
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10538382
• 关键词: Affinity; Alzheimer' s Disease; Autoradiography; Behavior; Binding; Biochemical; Biological Assay; Biomedical Research; Catalysis; Catalytic DNA; Cations; Characteristics; Chemistry; Computer Models; Crystallization; DNA; Deoxyribose; Detection; Dialysis procedure; Disease; Electron Spin Resonance Spectroscopy; Employment; Environment; Equilibrium; Foundations; Freezing; Future; Geometry; Goals; Healthcare; High Pressure Liquid Chromatography; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Investigation; Ions; Isotope Labeling; Kinetics; Knowledge; Literature; Location; Metal Binding Site; Metalloproteins; Metals; Methodology; Methods; Modeling; Modification; Molecular; Mossbauer Spectroscopy; Nature; Oligonucleotides; Oxidation-Reduction; Pathology; Physiologic pulse; Physiological; Point Mutation; Proteins; RNA; Reaction; Research Training; Rest; Risk Factors; Role; Series; Site; Solid; Specificity; Structure; Structure-Activity Relationship; System; Techniques; Therapeutic; Time; Training; Work; absorption; analog; base; biomaterial compatibility; biophysical properties; design; electronic structure; geometric structure; high reward; high risk; improved; in vivo; insight; next generation; novel; novel strategies; oxidation; phosphodiester; rational design; screening; sensor; spatiotemporal; vibration

PROJECT SUMMARY/ABSTRACT DNAzymes represent one of the most recent classes of diverse, catalytically active biomolecules. However, despite their discovery >25 years ago and exceptional potential for broad analytical and therapeutic applications, our understanding of metallo-DNAzymes in terms of binding selectivity, structure, and catalytic mechanism still lags far behind that of metalloproteins. Although DNAzymes have already been developed as highly selective metal ion sensors, the lack of fundamental knowledge regarding metallo-DNAzyme function has precluded the application of rational design to enhance metal binding affinity and specificity. The goal of this project is to obtain unparalleled insight into the structure-function relationships of metal-binding DNAzymes specific for redox-active metal ions (RAMIs) with high physiological relevance (e.g., Fe2+, Fe2+, Cu+, and Cu+2), and in turn provide a foundation for the future rational design of DNAzymes. To achieve this goal, an array of biochemical and advanced biophysical characterization techniques will be employed and cross-correlated to determine the locations of metal binding, coordination environments, binding affinities and specificities, and reaction mechanisms for a series of Fe2+, Fe3+, Cu+, and Cu+2-specific DNAzymes. Metal-bound DNAzyme resting states will be generated using a series of “non-cleavable” substrates, which will prove fundamental in determining metal binding affinities, specificities, and key spectroscopic signatures using UV-Vis/nIR, EPR, and 57Fe Mössbauer spectroscopies. By additionally applying XAS, the rudimentary coordination environment and electronic structure will be determined. Single point mutations will be screened across suspected metal-binding regions of oligonucleotide sequences, and the corresponding cleavage efficiency and characteristic spectroscopic signatures will be tracked to narrow the assignment of metal-binding site. Further advanced characterization using vibrational and pulse EPR spectroscopies will be used together with selectively isotope-labeled residues to provide a precise assignment of metal binding location and coordination environment. All of this information will be matched by computational modeling of first coordination binding models using a DFT and ab initio approaches. Lastly, a high-risk/high-reward foray will be made to grow diffraction-quality crystals for holistic structural characterization. Beyond the resting state, the mechanism of DNA/RNA cleavage by these DNAzymes will be analyzed by a combined analysis of cleaved fragment ends and careful kinetic characterization. Where necessary, rapid quench flow and rapid freeze quench methods will be employed to trap and assess potential reaction intermediates. Achieving the above goals will greatly deepen our understanding of the structure and function of metal- binding sites in DNAzymes, shifting the paradigm of metalloprotein characterization methodology to include metallo-DNAzymes. These insights are crucial for rational design and computational modeling to be used effectively in producing the next generation of metal ion-sensing DNAzymes.

项目摘要/摘要 dnazymes代表了最近的潜水员，催化活性的生物分子之一。然而， 尽管它们发现> 25年前，并且具有广泛的分析和治疗应用的非凡潜力，但 我们在结合选择性，结构和催化机制方面对金属 - 脱氮的理解仍然 落后于金属蛋白的落后。尽管dnazymes已经被开发为高度选择性 金属离子传感器，缺乏有关金属dnazyme功能的基本知识已阻止 合理设计的应用以增强金属结合亲和力和特异性。该项目的目标是获得 对氧化还原活性特有的金属结合dnazymes的结构功能关系的无与伦比的洞察力 具有高物理相关性的金属离子（RAMIS）（例如Fe2+，Fe2+，Cu+和Cu+2），进而提供了一个 Dnazymes未来理性设计的基础。 为了实现这一目标，一系列生化和先进的生物物理表征技术将是 使用并交叉相关以确定金属结合，协调环境的位置，结合 一系列Fe2+，Fe3+，Cu+和Cu+2特异性dnazymes的亲和力和特异性以及反应机制。 金属结合的dnazyme静止状态将使用一系列“不可裂解”底物生成 证明使用金属结合亲和力，特异性和关键光谱特征的基础 UV-VIS/NIR，EPR和57feMössbauer光谱镜。另外应用XAS，基本 将确定协调环境和电子结构。将筛选单点突变 跨怀疑的寡核苷酸序列的金属结合区域以及相应的裂解 效率和特征性光谱特征将被跟踪以缩小金属结合的分配 地点。使用振动和脉搏EPR光谱镜将进一步的高级表征一起使用 具有选择性的同位素标记的结果，以提供金属结合位置的精确分配和 协调环境。所有这些信息都将通过第一次协调的计算建模匹配 使用DFT和AB始于方法的绑定模型。最后，将进行高风险/高奖金的尝试 衍射质量晶体，用于整体结构表征。超越静止状态，机制 这些dnazymes的DNA/RNA裂解将通过对切割片段末端和 精心的动力学表征。必要时，快速淬火流量和快速冻结方法将是 用于捕获和评估潜在反应中间体。 实现上述目标将大大加深我们对金属的结构和功能的理解 dnazymes中的结合位点，将金属蛋白表征方法的范式转移到包括 金属豆氮。这些见解对于使用理性设计和计算建模至关重要 有效地产生了下一代金属离子感应dnazymes。