Design of Intrasterically Regulated Chimeric Enzymes

描述(由申请者提供)：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理解来彻底改变科学和医学。随着我们对各种人类疾病潜在生化原因的基本知识的迅速提高，个性化治疗、治疗过程中对患者的更好监控以及护理点诊断将越来越受到重视。这些新兴领域的进展将依赖于便携式、易于使用、可以显示出高检测灵敏度、准确性、分析速度和吞吐量的生物传感器技术。在这里，我们提出了一个生物分子传感的基础研究计划，该计划的灵感来自于空间内调节的天然酶的功能物理化学原理。所提出的生物传感器元件在设计上是模块化的，并且基于酶通过变构系链与其小分子抑制剂的特定共价结合。当系绳与其补体(核酸、多肽或蛋白质)相互作用时，随后的构象变化通过将抑制物从其活性部位释放出来来激活酶。激活的酶复合体通过底物周转，充当分子识别事件的内置信号放大器。这一新概念的实用性已经在高灵敏度的一步检测特定DNA序列的设计中得到了确立(初步结果部分)。拟议的研究计划旨在进一步推进和优化这一新的传感方法。提出的研究计划的具体目标是：1.通过对抑制剂的酶结合部位、DNA系链结构和大小以及抑制剂的共价结构的合理修饰，设计和表征抑制物-DNA-酶(IDE)嵌合体的改良变体。设计用于快速检测生物相关遗传标记和单核苷酸多态(SNP)的IDE。通过设计识别HIV病毒中GP-41的抑制剂-多肽-酶(IPE)嵌合体，探索空间内调控酶嵌合体在蛋白质检测中的共性。

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.