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

基本信息

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
项目类别：
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

项目摘要

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，我们计划实现对复杂异核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）、血红素的结构特征铜氧化酶 (HCO) 和亚硫酸盐还原酶 (SiR) 负责高效且选择性的 2-、4- 和 6 电子分别催化还原NO、O2和SO32-，尽管研究已经取得了很大进展。对于单个酶，我们知识的一个主要差距是哪些结构特征造成了差异为了填补这一空白，我们计划使用小而稳定的蛋白质作为“支架”来制造“生物合成”。通过将不同的血红素-非血红素金属离子放入具有类似高活性的天然酶的模型中。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；和 d) 研究血红素辅助因子在 HCO、NOR 和 SiR 中的作用实现这一目标将提供对金属蛋白结构、功能和设计的更深入的了解。对生物催化具有广泛的影响，可以设计用于生化和生物医学应用的生物催化剂。在第二个项目中，我们计划通过氧化选择对不同金属离子具有高选择性的DNAzymes 陈述特异性并探索这些 DNAzyme 作为顺磁性金属离子成像剂的应用（PMI），例如活体中的 Fe 及其 Fe2+/Fe3+ 氧化还原循环，同时在开发方面也取得了进展。对于金属离子的传感器，能够选择性检测PMI的传感器很少（如果有的话）；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+ 的成像使用催化信标在活细胞中同时检测 Fe3+ 我们计划开发时空控制方法。基于 DNAzyme 的成像和 DNAzyme 的细胞内生成，以探索其成像应用。实现这一目标将更深入地了解 PMI 及其氧化还原循环在诸如铁死亡与神经退行性疾病和细菌感染有关。最后，在高风险和高回报的努力中，我们建议将 DNAzyme 的应用扩展为新的用于切割双链 DNA (dsDNA) 和基因组编辑的基因工程工具，作为替代品为了实现这一目标，我们计划开发新的限制性内切酶和 CRISPR/Cas。肽核酸辅助DNAzymes进行双链DNA切割，然后建立细胞内基因编辑实现这一目标将允许更小、更强大的DNA酶用于高度定制的重组。 DNA 克隆和高保真基因组编辑。