Protease chain reactions for molecular analysis of cancer markers

用于癌症标志物分子分析的蛋白酶链反应

• 批准号: 8222417
• 负责人: Biao Ruan
• 金额: $ 15万
• 依托单位: POTOMAC AFFINITY PROTEINS, LLC
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-19 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8222417
• 关键词: Affinity; Analog Computers; Antibodies; Bacillus (bacterium); Binding; Biological; Biological Assay; Cancer Detection; Characteristics; Cleaved cell; Complex; DNA amplification; Detection; Development; Elements; Engineering; Enzyme-Linked Immunosorbent Assay; Enzymes; Funding; Goals; Grant; Link; Logic; Malignant Neoplasms; Marketing; Measures; Methods; Molecular; Molecular Analysis; Output; Peptide Hydrolases; Phase; Polymerase Chain Reaction; Protease Inhibitor; Proteins; Reaction; Resources; Sampling; Signal Transduction; Site; Small Business Innovation Research Grant; Subtilisins; System; Technology; Time; United States National Institutes of Health; antibody conjugate; cancer prevention; design and construction; improved; inhibitor/antagonist; molecular marker; nano; novel; prototype; technological innovation

DESCRIPTION (provided by applicant): Our objective is to develop a protease chain reaction technology (ProCR) which will enable ultra- sensitive molecular detection. A protease chain reaction has four basic components: 1) a protease conjugated to a binding molecule, 2) an unconjugated protease, 3) an inhibitor protein which contains a proteolytic cleavage site and 4) a protease substrate which generates a signal upon its cleavage. Fundamentally, a protein chain reaction is a powerful analogue computer with two key characteristics which greatly facilitate the detection of target molecules. 1) It can convert the concentration of a specific target molecule into a time signature. 2) It can create enormous signal amplification, analogous to the amplification of DNA by a polymerase chain reaction (PCR). Thus detection is enabled because the final observable signal produced by a target molecule can be very large and the time lag until onset of the signal is precisely correlated with the concentration of target molecule. The critical elements for controlling the protease chain reaction (and ultimately determining the sensitivity of assays linked to it) are very tight inhibition of the protease by the intact inhibitor and cleavage of the inhibitor by free protease. Accordingly, the three experimental Aims are: 1) Engineering the subtilisin prodomain to maximize inhibition; 2) Design and construct prodomain inhibitors with cleavable loops; 3) Construct and characterize a prototype chain reaction useful for detection. The Phase I goal is to demonstrate proof of principle for protease chain reactions and their applicability for molecular detection. The three milestones are: 1) Develop tight inhibitors with KI d 100pM; 2) Engineer cleavable loops into the inhibitors of Aim 1 which are cleaved a rate > 1000 M-1s-1; 3) Detect 1 femtomole of protease- conjugated antibody by ProCR in a microtiter dish assay. The long term goal is to develop protease- inhibitor complexes as enzymatic nano-processors which can be combined to detect multiple signals and to control output with multiple logic gates. PUBLIC HEALTH RELEVANCE: The development of polymerase chain reaction (PCR) technology demonstrated the extraordinary power of harnessing an enzyme to perform novel, programmable reactions. Our objective is here to develop an analogous protease chain reaction technology (ProCR) which will enable ultra-sensitive molecular detection. The long term goal is to improve cancer detection and prevention by enabling accurate quantitation of multiple, low abundance molecular markers.

描述(申请人提供)：我们的目标是开发一种能够进行超灵敏分子检测的蛋白酶链式反应技术(PROR)。蛋白酶链式反应有四个基本成分：1)与结合分子结合的蛋白酶，2)非结合的蛋白酶，3)包含蛋白水解性裂解位点的抑制蛋白，以及4)在其裂解时产生信号的蛋白酶底物。从根本上说，蛋白质链式反应是一台功能强大的模拟计算机，具有两个关键特征，极大地促进了目标分子的检测。1)它可以将特定目标分子的浓度转换为时间信号。2)它可以产生巨大的信号放大，类似于通过聚合酶链式反应(PCR)扩增DNA。因此，检测是可能的，因为目标分子产生的最终可观察信号可能非常大，并且信号开始之前的时间延迟与目标分子的浓度精确相关。控制蛋白酶链式反应(并最终决定与之相关联的检测方法的灵敏度)的关键因素是完整的抑制物对蛋白酶的严格抑制和游离酶对抑制物的切割。因此，三个实验目标是：1)设计枯草杆菌蛋白原结构域以最大限度地抑制作用；2)设计和构建具有可切割环的原结构域抑制剂；3)构建和表征对检测有用的原型链式反应。第一阶段的目标是证明蛋白酶链式反应的原理及其在分子检测中的适用性。这三个里程碑是：1)开发Ki d为100pM的紧密抑制剂；2)将可切割的环工程到Aim 1的抑制剂中，并以1000M-1s-1的速度切割；3)在微量滴度盘法中用PROCR检测1个Femtomole的蛋白酶结合抗体。其长期目标是开发酶抑制复合体作为酶纳米处理器，它可以组合在一起检测多个信号，并用多个逻辑门控制输出。 公共卫生相关性：聚合酶链式反应(PCR)技术的发展展示了利用一种酶进行新颖的、可编程的反应的非凡力量。我们的目标是开发一种类似的蛋白酶链式反应技术(PROR)，使超灵敏分子检测成为可能。长期目标是通过准确定量多个低丰度分子标记来改进癌症检测和预防。