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.

项目总结/摘要 DNA酶代表最新类别的多样的催化活性生物分子之一。然而，在这方面， 尽管它们是在25年前发现的并且具有广泛的分析和治疗应用的特殊潜力， 我们对金属-DNA酶在结合选择性、结构和催化机制方面的理解仍然是 远远落后于金属蛋白。尽管DNA酶已经被开发成具有高度选择性， 金属离子传感器，缺乏关于金属-DNA酶功能的基础知识， 应用合理设计以增强金属结合亲和力和特异性。该项目的目标是获得 对金属结合DNA酶的结构-功能关系的无与伦比的洞察力， 具有高生理相关性的金属离子（RAMI）（例如，Fe 2+、Fe 2+、Cu+和Cu+2），并进而提供 为今后合理设计DNA酶奠定了基础。 为了实现这一目标，一系列生物化学和先进的生物物理表征技术将被 采用和交叉相关，以确定金属结合的位置，配位环境，结合 一系列Fe ~（2+）、Fe ~（3+）、Cu ~（2+）和Cu ~（2+）特异性DNA酶的亲和力和特异性以及反应机理。 金属结合的DNA酶静止状态将使用一系列“不可切割”底物产生，其将 证明在确定金属结合亲和力，特异性和关键光谱特征的基础上， UV-Vis/nIR、EPR和57 Fe穆斯堡尔谱。通过另外应用XAS， 确定协调环境和电子结构。将筛选单点突变 跨越寡核苷酸序列的可疑金属结合区域， 效率和特征光谱签名将被跟踪，以缩小金属结合的分配 绝佳的价钱使用振动和脉冲EPR光谱的进一步先进表征将一起使用 用选择性同位素标记的残基提供金属结合位置的精确分配， 协调环境。所有这些信息将通过第一协调的计算建模来匹配 结合模型使用DFT和从头算方法。最后，将进行高风险/高回报的尝试， 用于整体结构表征的衍射质量晶体。除了休息状态， 这些DNA酶的DNA/RNA切割将通过切割片段末端的组合分析来分析， 仔细的动力学表征。必要时，将采用快速淬火流和快速冷冻淬火方法。 用于捕获和评估潜在的反应中间体。 实现上述目标将大大加深我们对金属结构和功能的认识， 结合位点的DNA酶，转移范式的金属蛋白表征方法，包括 金属DNA酶这些见解是至关重要的合理设计和计算建模使用 有效地生产下一代金属离子传感DNA酶。