Design of Intrasterically Regulated Chimeric Enzymes

空间调控嵌合酶的设计

• 批准号: 6571379
• 负责人: M. Reza Ghadiri
• 金额: $ 35.59万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6571379
• 关键词: HIV envelope protein gp120; HIV envelope protein gp41; active sites; allosteric site; binding sites; biosensor device; carboxypeptidase; chemical synthesis; enzyme activity; gel electrophoresis; gel filtration chromatography; genetic markers; genetic polymorphism; human immunodeficiency virus; nucleic acid quantitation /detection; nucleic acid sequence; physical chemical interaction; plasmids; protein protein interaction; protein structure function; technology /technique development; tissue mosaicism

DESCRIPTION (provided by applicant): The post genomic era promises to revolutionize science and medicine through a detailed understanding of biology at the molecular and system levels. As our fundamental knowledge of the underlying biochemical causes of various human ailments rapidly improves, an ever increasing emphasis will be placed on personalized treatments, better monitoring of patients during the course of therapy, and point-of-care diagnostics. Advances in these emerging fields will depend on biosensor technologies that are portable, easy to use, and can display high detection sensitivity, accuracy, analysis speed, and throughput. Here we propose a basic research program in biomolecular sensing that is inspired by the functioning physicochemical principles of intrasterically regulated natural enzymes. The proposed biosensor element is modular in design and is based on specific covalent attachment of an enzyme to its small molecule inhibitor through an allosteric tether. Upon interaction of the tether with its complement (nucleic acid, peptide, or protein), the ensuing conformational change activates the enzyme by liberating the inhibitor from its active site. The activated enzyme complex, through substrate turnover, serves as a built-in signal amplifier of the molecular recognition event. The utility of this novel concept has already been established in the design of a highly sensitive one step detection of specific DNA sequences (Preliminary Results section). The proposed research program seeks to further advance and optimize this new sensing methodology. The specific aims of the proposed research program are: I. To design and characterize improved variants of the Inhibitor-DNA-Enzyme (IDE) chimera through rational modifications of the enzyme attachment site, DNA tether structure and size, and the covalent structure of the inhibitor. II. To design IDEs for rapid detection of biologically relevant genetic markers and single nucleotide polymorphism (SNP). III. To explore the generality of the intrasterically regulated enzyme chimera in protein detection by engineering an Inhibitor-Peptide-Enzyme (IPE) chimera for recognition of GP-41 in the HIV virus.

描述（由申请人提供）：后基因组时代承诺通过在分子和系统水平上对生物学的详细了解来彻底改变科学和医学。随着我们对各种人类疾病的潜在生化原因的基本知识迅速提高，个性化治疗、治疗过程中对患者的更好监测以及即时诊断将得到越来越多的重视。这些新兴领域的进步将依赖于便携式、易于使用的生物传感器技术，并且可以显示高检测灵敏度、准确性、分析速度和吞吐量。在此，我们提出了一个生物分子传感的基础研究计划，该计划受到肠内调节天然酶的功能物理化学原理的启发。所提出的生物传感器元件是模块化设计的，并且基于酶通过变构系绳与其小分子抑制剂的特定共价附着。当链链与其补体（核酸、肽或蛋白质）相互作用时，随后的构象变化通过从活性位点释放抑制剂来激活酶。激活的酶复合体，通过底物翻转，作为分子识别事件的内置信号放大器。这个新概念的实用性已经在设计一个高灵敏度的一步检测特定的DNA序列（初步结果部分）。提出的研究计划旨在进一步推进和优化这种新的传感方法。提出的研究计划的具体目标是：1 .通过合理修饰酶的附着位点、DNA链的结构和大小以及抑制剂的共价结构，设计和表征抑制剂-DNA-酶（IDE）嵌合体的改进变体。2。目的：设计用于生物相关遗传标记和单核苷酸多态性（SNP）快速检测的ide。3。通过构建抑制肽酶（IPE）嵌合体识别HIV病毒中的GP-41，探讨腹腔内调节酶嵌合体在蛋白质检测中的普遍性。