Protease chain reactions for molecular analysis of cancer markers

描述（由申请人提供）：我们的目标是开发一种蛋白酶链反应技术（ProCR），该技术将实现超灵敏的分子检测。蛋白酶链式反应有四个基本组成部分：1)与结合分子偶联的蛋白酶，2)未偶联的蛋白酶，3)含有水解裂解位点的抑制蛋白，4)在裂解过程中产生信号的蛋白酶底物。从根本上说，蛋白质链式反应是一个强大的模拟计算机，具有两个关键特性，极大地促进了目标分子的检测。1)它可以将特定靶分子的浓度转化为时间特征。2)它可以产生巨大的信号扩增，类似于通过聚合酶链反应（PCR）扩增DNA。因此，检测是可行的，因为目标分子产生的最终可观察信号可能非常大，并且直到信号开始的时间滞后与目标分子的浓度精确相关。控制蛋白酶链式反应的关键因素（并最终决定与之相关的测定的敏感性）是完整抑制剂对蛋白酶的非常紧密的抑制和游离蛋白酶对抑制剂的裂解。因此，实验的三个目标是：1)对枯草杆菌素原域进行工程化，使抑制作用最大化；2)设计构建具有可切割环的原结构域抑制剂；3)构建和表征一个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反应的原理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)在Aim 1抑制剂中植入可切割环，其切割速率为1000 M-1s-1；3)微滴皿法，用ProCR检测1飞莫蛋白酶偶联抗体。长期目标是开发蛋白酶抑制剂复合物作为酶的纳米处理器，它可以结合起来检测多个信号并通过多个逻辑门控制输出。

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.