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
• 负责人: Yi Lu
• 金额: $ 58.13万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-16 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415131
• 关键词: Address; Affinity; Bacterial Infections; Binding; Biochemical; Biological; Biological Process; Catalytic DNA; Cells; Cloning; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Diagnostic Imaging; Electrons; Engineering; Enzymes; Exhibits; Generations; Genes; Genetic Engineering; Goals; Health; Heart; Heme; Image; In Vitro; Individual; Ions; Knowledge; Metalloproteins; Metals; Methods; Modeling; Neurodegenerative Disorders; Nitrogen; Organism; Oxidation-Reduction; Peptide Nucleic Acids; Process; Proteins; Reaction; Recombinant DNA; Respiration; Role; Scaffolding Protein; Specificity; Structure; Structure-Activity Relationship; Sulfite reductase; Sulfur; Time; Tyrosine; base; bioimaging; biological systems; cofactor; copper oxidase; design; ds-DNA; functional mimics; genome editing; heme a; high risk; imaging agent; insight; metalloenzyme; nitric oxide reductase; novel; oxidation; restriction enzyme; scaffold; sensor; spatiotemporal; tool

Project summary/Abstract The overall goal is to design and select two classes of metalloenzymes, metalloprotein enzymes and metallo- DNAzymes, and to explore their applications in biocatalysis, bioimaging, and genetic engineering. In the first project, we plan to achieve a holistic understanding of complex heteronuclear metalloenzymes involved in multi-electron processes, specifically structural features in nitric oxide reductases (NOR), heme- copper oxidases (HCO) and sulfite reductases (SiR) responsible for efficient and selective 2-, 4-, and 6 electron catalytic reduction of NO, O2, and SO32-, respectively. Even though much progress has been made in studying individual enzymes, a major gap in our knowledge is what structural features are responsible for the differences in their functions. To fill this gap, we plan to use small and stable proteins as “scaffolds” to make “biosynthetic models” of native enzymes with similarly high activity. By placing different heme–nonheme metal ions into the same protein scaffold, we plan to a) understand how a heme-Cu center can exhibit either HCO or SiR activity; b) elucidate structural features responsible for catalytic activity and substrate binding affinity in SiR; c) clarify the roles of tyrosine in HCO and SiR activities; and d) investigate roles of heme cofactors in HCO, NOR, and SiR activities. Accomplishing this goal will offer deeper insight into metalloprotein structure, function, and design, and have a broad impact on biocatalysis, allowing design of biocatalysts for biochemical and biomedical applications. In the second project, we plan to select DNAzymes with high selectivity for different metal ions with oxidation state specificity and explore applications of these DNAzymes as imaging agents for paramagnetic metal ions (PMIs) such as Fe and its Fe2+/Fe3+ redox cycle in living organisms. While progress has been made in developing sensors for metal ions, sensors that can selectively detect PMIs are limited; few, if any, can detect two oxidation states of the same metal ions simultaneously. To overcome this barrier, we have obtained DNAzymes sensors with high selectivity for either Fe2+ or Fe3+ using in vitro selection and demonstrated imaging of both Fe2+ and Fe3+ simultaneously in living cells using catalytic beacons. We plan to develop methods for spatiotemporal control of DNAzyme-based imaging and for intracellular generation of DNAzymes to explore their imaging applications. Accomplishing this goal will offer deeper insight into the roles of PMIs and their redox cycles in processes such as ferroptosis that has been associated with neurodegenerative diseases and bacterial infections. Finally, in a high-risk and high-return endeavor, we propose to expand DNAzyme’s applications as new genetic engineering tools for cleaving double-stranded DNA (dsDNA) and for genome editing, as alternatives to protein restriction enzymes and CRISPR/Cas, respectively. To achieve the goal, we plan to develop novel peptide nucleic acid-assisted DNAzymes for dsDNA cleavage and then establish an intracellular gene-editing platform. Achieving this goal will allow smaller and more robust DNAzymes for highly customizable recombinant DNA cloning and high-fidelity genome editing.

项目概要/摘要 总体目标是设计和选择两类金属酶，金属蛋白酶和金属蛋白酶。 DNA酶，并探讨其在生物催化，生物成像和基因工程中的应用。 在第一个项目中，我们计划实现对复杂的杂合金属酶的整体理解 参与多电子过程，特别是一氧化氮还原酶（NOR），血红素， 铜氧化酶（HCO）和亚硫酸盐还原酶（SiR）负责有效和选择性的2-，4-和6电子 分别催化还原NO、O2和SO 32-。尽管在研究方面取得了很大进展， 对于单个酶，我们知识中的一个主要空白是什么结构特征是造成差异的原因 在他们的职能。为了填补这一空白，我们计划使用小而稳定的蛋白质作为“支架”， 具有类似高活性的天然酶的“模型”。通过将不同的血红素-非血红素金属离子放入 同样的蛋白质支架，我们计划a）了解血红素-Cu中心如何表现出HCO或SiR活性; B）阐明SiR中负责催化活性和底物结合亲和力的结构特征; 酪氨酸在HCO和SiR活性中的作用;以及d）研究血红素辅因子在HCO、NOR和SiR中的作用 活动实现这一目标将提供更深入的了解金属蛋白质的结构，功能和设计， 对生物催化具有广泛的影响，允许设计用于生物化学和生物医学应用的生物催化剂。 在第二个项目中，我们计划选择对不同金属离子具有高选择性的DNA酶 这些DNA酶状态特异性及其作为顺磁性金属离子显像剂的应用 （PMIs）如Fe及其在活生物体中的Fe 2 +/Fe 3+氧化还原循环。虽然在发展方面取得了进展， 用于金属离子的传感器，可以选择性地检测PMI的传感器是有限的;很少，如果有的话，可以检测两种氧化 金属离子的同时。为了克服这一障碍，我们获得了DNA酶传感器， 使用体外选择对Fe 2+或Fe 3+具有高选择性，并证明了Fe 2+和 Fe 3+同时在活细胞中使用催化信标。我们计划开发时空控制方法 的DNA酶为基础的成像和细胞内产生的DNA酶，探索其成像应用。 实现这一目标将提供更深入的了解PMI及其氧化还原循环在过程中的作用， 如与神经退行性疾病和细菌感染相关的铁下垂症。 最后，在一个高风险和高回报的奋进，我们建议扩大DNAzyme的应用，作为新的 用于切割双链DNA（dsDNA）和用于基因组编辑的基因工程工具，作为 蛋白质限制酶和CRISPR/Cas。为了实现这一目标，我们计划开发新的 肽核酸辅助DNA酶切割dsDNA，然后建立细胞内基因编辑 平台实现这一目标将允许更小和更强大的DNA酶用于高度可定制的重组 DNA克隆和高保真基因组编辑。