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: 10476760
• 负责人: Yi Lu
• 金额: $ 34.46万
• 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/10476760
• 关键词: Design; Selection; Novel; Metalloenzymes; Biocatalysis

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 总的目标是设计和选择两类金属酶，金属蛋白酶和金属-蛋白质酶。 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的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)的结构特征，血红素- 铜氧化酶(HCO)和亚硫酸盐还原酶(SIR)负责高效和选择性的2-，4-和6电子 NO、O2和SO32-的催化还原。尽管在研究方面取得了很大的进步 单个酶，我们知识中的一个主要差距是什么结构特征导致了差异 在他们的职能中。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” 具有类似高活性的原生酶的模型。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-铜中心如何表现出HCO或SIR活性； B)阐明SIR中催化活性和底物结合亲和力的结构特征；c)澄清 酪氨酸在HCO和SIR活性中的作用；以及d)调查血红素辅助因子在HCO、NOR和SIR中的作用 活动。实现这一目标将提供对金属蛋白结构、功能和设计的更深层次的了解，以及 对生物催化有广泛的影响，允许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，我们计划选择对不同金属离子具有高选择性的氧化脱氧核酶 这些DNAzyme作为顺磁性金属离子显像剂的状态特异性及其应用探讨 (PMIs)，如铁及其在生物体内的Fe2+/Fe3+氧化还原循环。在发展方面取得进展的同时， 金属离子的传感器，能够选择性地检测PMI的传感器是有限的；如果有的话，很少人能检测到两种氧化 相同金属离子的同时状态。为了克服这个障碍，我们已经获得了DNAzymes传感器 具有对Fe2+或Fe3+的高选择性，使用体外选择和展示Fe2+和Fe3+两者的成像 利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的方法 用于基于DNAzyme的成像和细胞内DNAzyme的生成，以探索其成像应用。 实现这一目标将使我们更深入地了解PMI及其氧化还原周期在 与神经退行性疾病和细菌感染有关的铁性下垂。 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 切割双链DNA(DsDNA)和基因组编辑的基因工程工具，作为 蛋白质限制性内切酶和CRISPR/Cas。为了实现这一目标，我们计划开发小说 多肽核酸辅助DNAzyme对dsDNA的切割并建立细胞内基因编辑 站台。实现这一目标将允许更小和更强大的DNAzyme用于高度定制的重组 DNA克隆和高保真基因组编辑。