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

项目摘要/摘要 DNAzyme代表了一类最新的具有催化活性的多样性生物分子。然而， 尽管它们是在25年前发现的，而且在广泛的分析和治疗应用方面具有非凡的潜力。 我们对金属脱氧核酶在结合选择性、结构和催化机理方面的理解仍然 远远落后于金属蛋白。尽管DNA酶已经被开发为高度选择性的 对于金属离子传感器，由于缺乏关于金属脱氧核酶功能的基础知识， 应用合理设计，增强金属结合亲和力和特异性。这个项目的目标是获得 对氧化还原活性特异的金属结合DNAzyme结构-功能关系的无与伦比的洞察 具有高度生理相关性的金属离子(Ramis)(例如，Fe2+、Fe2+、Cu2+和Cu2+)，进而提供 为未来DNA酶的合理设计奠定了基础。 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 采用并相互关联来确定金属结合、配位环境、结合 一系列Fe2+、Fe3+、Cu2+和Cu2+特异性DNA酶的亲和力和特异性以及反应机理。 金属结合的DNAzyme休眠状态将使用一系列“不可切割”的底物来产生，这将 证明是确定金属结合亲和力、特异性和关键光谱特征的基础 UV-Vis/NIR、EPR和57Fe穆斯堡尔光谱。通过额外应用XAS，基本的 将确定配位环境和电子结构。将筛查单点突变 跨越寡核苷酸序列的可疑金属结合区，以及相应的切割 将跟踪效率和特征光谱特征，以缩小金属结合的分配范围 地点。使用振动和脉冲EPR波谱的进一步高级表征将一起使用 具有选择性的同位素标记残基以提供金属结合位置的精确分配以及 协调环境。所有这些信息都将通过第一个坐标的计算模型进行匹配 使用DFT和从头算方法绑定模型。最后，将进行一次高风险/高回报的进军以实现增长 用于整体结构表征的衍射级晶体。在静止状态之外， 这些DNAzyme对DNA/RNA的裂解将通过裂解片段末端和 仔细的运动学表征。在必要时，将采用快速冷却流动和快速冻结冷却方法 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-- DNAzyme中的结合位点，改变了金属蛋白表征方法的范式，以包括 金属脱氧核酶。这些见解对于使用合理的设计和计算建模至关重要 有效地生产下一代金属离子敏感DNAzyme。

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.