Protease chain reactions for molecular analysis of cancer markers

总结 我们的目标是开发一种蛋白酶链式反应技术（ProCR）， 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 缀合至结合分子，2）未缀合的蛋白酶，3）抑制剂蛋白，其含有 蛋白水解切割位点和4）在其切割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个强大的模拟计算机，具有两个关键特征 这极大地促进了目标分子的检测。1)它可以将特定的 将目标分子转化为时间标记。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，能够进行检测，因为 由靶分子产生的最终可观察信号可能非常大，并且直到开始的时间滞后 该信号与靶分子的浓度精确相关。关键要素 控制蛋白酶链式反应（并最终确定与之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和游离的抑制剂对蛋白酶的切割 蛋白酶因此，三个实验目的是：1）工程化枯草杆菌蛋白酶前结构域， 最大化抑制; 2）设计和构建具有可切割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 蛋白酶链式反应原理的证明及其在分子检测中的应用。三 里程碑是：1）开发具有KI d 100 pM的紧密抑制剂; 2）将可裂解环工程化到 切割速率> 1000 M-1 s-1的Aim 1抑制剂; 3）检测1飞摩尔的蛋白酶， 在微量滴定皿测定中通过ProCR测定缀合的抗体。长期目标是开发蛋白酶- 作为酶纳米处理器的抑制剂复合物，其可以组合以检测多种信号 并且用多个逻辑门控制输出。

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.