Design of Intrasterically Regulated Chimeric Enzymes

超出所提供的空间。后基因组时代有望通过在分子和系统水平上对生物学的详细理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础知识迅速提高，越来越多的重点将放在个性化治疗，治疗过程中更好地监测患者以及即时诊断上。这些新兴领域的进展将取决于便携式，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，分析速度和通量。在这里，我们提出了一个生物分子传感的基础研究计划，其灵感来自于内部调控的天然酶的功能物理化学原理。所提出的生物传感器元件是模块化设计的，并且是基于酶通过变构系链与其小分子抑制剂的特异性共价连接。在系链与其互补物（核酸、肽或蛋白质）相互作用后，随后的构象变化通过将抑制剂从其活性位点释放而激活酶。活化的酶复合物，通过底物周转，作为一个内置的信号放大器的分子识别事件。这种新概念的实用性已经在设计特异性DNA序列的高灵敏度一步检测中得到确立（初步结果部分）。拟议的研究计划旨在进一步推进和优化这种新的传感方法。拟议研究计划的具体目标是：一。通过合理修饰酶连接位点、DNA系链结构和大小以及抑制剂的共价结构，设计并表征抑制剂-DNA-酶（IDE）嵌合体的改良变体。二.设计IDES用于快速检测生物学相关的遗传标记和单核苷酸多态性（SNP）。三.通过构建识别HIV病毒GP-41的抑制肽-酶（IPE）嵌合体，探索层内调控酶嵌合体在蛋白质检测中的通用性。性能现场=

EXCEED THE SPACE PROVIDED. 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. PERFORMANCE SITE ========================================Section End===========================================