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; 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.

项目摘要/摘要 总体目标是设计和选择两类的金属酶，金属蛋白酶和金属酶 - dnazymes，并探索其在生物催化，生物成像和基因工程中的应用。 在第一个项目中，我们计划对复杂的异核金属酶进行整体理解 参与多电子过程，特别是一氧化氮减少（NOR）的结构特征，血红素 铜氧化物（HCO）和亚硫酸盐减少了（SIR），负责有效和选择性2-，4-和6电子 NO，O2和SO32-的催化减少。即使在学习方面取得了很多进展 单个酶，我们所知的主要差距是结构特征是造成差异的原因 在他们的功能中。为了填补这一空白，我们计划将小而稳定的蛋白质用作“脚手架”来制作“生物合成 具有类似活性的天然酶的模型”。通过将不同的血红素金属离子置于 相同的蛋白质支架，我们计划a）了解Heme-Cu中心如何表现HCO或SIR活动； b）阐明负责催化活性和底物结合亲和力的结构特征； c）澄清一下 酪氨酸在HCO和爵士活动中的作用； d）研究血红素辅因子在HCO中的作用 活动。实现这一目标将为金属蛋白结构，功能和设计提供更深入的了解，并且 对生物催化有广泛的影响，从而设计生物化学和生物医学应用的生物催化剂。 在第二个项目中，我们计划选择具有氧化不同金属离子的高选择性的dnazymes 状态特异性和探索这些dnazymes作为顺磁金属离子成像剂的应用 （PMI）例如，在活生物体中的Fe及其Fe2+/Fe3+氧化还原周期。虽然在发展方面取得了进展 金属离子的传感器，可以选择性检测PMI的传感器受到限制；很少有（如果有的话）可以检测到两个氧化 同一金属离子的状态。为了克服这一障碍，我们获得了Dnazymes传感器 使用体外选择对Fe2+或Fe3+具有高选择性，并证明了Fe2+和 Fe3+仅在活细胞中使用催化信标。我们计划开发时空控制的方法 基于Dnazyme的成像和细胞内生成Dnazymes探索其成像应用。 实现这一目标将为PMI及其在此类过程中的氧化还原周期的作用提供更深入的了解 作为与神经退行性疾病和细菌感染有关的铁铁作用。 最后，在一项高风险和高回报的努力中，我们建议将Dnazyme的应用扩大为新的应用 用于切割双链DNA（DSDNA）和基因组编辑的基因工程工具，作为替代品 蛋白质限制酶和CRISPR/CAS。为了实现目标，我们计划开发小说 肽核酸辅助的dnazymes进行DsDNA裂解，然后建立细胞内基因编辑 平台。实现这一目标将允许更小，更健壮的dnazymes进行高度定制的重组 DNA克隆和高保真基因组编辑。