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: 8334483
• 负责人: Biao Ruan
• 金额: $ 15.16万
• 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/8334483
• 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

SUMMARY 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.

概括 我们的目标是开发一种蛋白酶链式反应技术 (ProCR)，该技术将使超 灵敏的分子检测。蛋白酶链式反应有四个基本组成部分：1) 蛋白酶 与结合分子缀合，2) 未缀合的蛋白酶，3) 包含 蛋白水解切割位点和4) 蛋白酶底物，其在切割时产生信号。 从根本上讲，蛋白质链式反应是一种功能强大的模拟计算机，具有两个关键特征 极大地方便了目标分子的检测。 1）可换算特定浓度 目标分子转化为拍号。 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。因此启用检测是因为 目标分子产生的最终可观察信号可能非常大，并且直到信号开始出现的时间滞后 信号与目标分子的浓度精确相关。的关键要素 控制蛋白酶链式反应（并最终确定与其相关的测定的灵敏度） 完整的抑制剂对蛋白酶的非常紧密的抑制以及游离的抑制剂对蛋白酶的裂解 蛋白酶。因此，三个实验目标是：1) 将枯草杆菌蛋白酶前结构域改造为 最大限度地抑制； 2) 设计并构建具有可裂解环的前结构域抑制剂； 3）构建 并表征可用于检测的原型链式反应。第一阶段的目标是证明 蛋白酶链式反应的原理证明及其在分子检测中的适用性。三个 里程碑是： 1) 开发 KI d 100pM 的紧密抑制剂； 2) 将可切割环设计成 目标 1 的抑制剂，其裂解率 > 1000 M-1s-1； 3) 检测1飞摩尔蛋白酶- 在微量滴定盘测定中通过 ProCR 结合抗体。长期目标是开发蛋白酶 抑制剂复合物作为酶纳米处理器，可以组合起来检测多个信号 并用多个逻辑门控制输出。