MOLECULAR BEACONS FOR PROTEIN DETECTION

描述(由申请者提供)：以高度多元化的方式快速检测和定量特定蛋白质的能力对基础科学研究、药物研究非常重要 筛选应用程序和临床诊断。在这一应用中，我们建议开发一种新的方法，它将允许生成能够同时识别特定蛋白质分子并发出识别信号的报告分子。由这些“分子信标”产生的信号将很容易测量荧光强度，这将 促进这一新方法的高吞吐量使用。建议的方法将允许设计真正的均一化验。这种方法学最初将针对具有自然序列特异性DNA结合活性的蛋白质而开发。随后，这一方法将扩展到包括不具有自然序列特异性核酸结合活性的蛋白质。这个项目将分两个阶段进行。在第一阶段，我们将使用模型蛋白质系统来提供“原则证明”证据，证明我们的方法可以用于DNA结合蛋白质，并且它可以扩展到缺乏这种活性的蛋白质。在项目的第二阶段，我们将把项目第一阶段提出的概念应用于 产生识别四种癌症相关靶点的“分子灯塔”：p53--一种肿瘤抑制因子，其失活是癌细胞中最常见的缺陷；核因子-kB-转录因子，参与许多基因的调节并被发现在许多肿瘤中具有结构性活性；p16(INK4A)--一种参与细胞周期调节的肿瘤抑制因子，其突变已被 在70多种不同类型的肿瘤细胞中被发现，以及p27(Kip1)-一种细胞周期抑制物，其细胞水平被证明是乳腺癌患者的重要预后标志。我们预计，我们建议开发的方法学将在基础癌症研究、疾病的分子诊断、治疗标志物的鉴定和 目标，以及对药物反应的特征。

DESCRIPTION (provided by applicant):The ability to rapidly detect and quantify specific proteins in a highly multiplexed manner is of great importance for basic science research, drug screening applications, and clinical diagnosis. In this application we propose the development of a novel methodology which will allow generation of reporter molecules capable at the same time to recognize and to signal the recognition of a specific protein molecule. The signal generated by these "molecular beacons" will be simple to measure fluorescence intensity, which will facilitate high-throughput use of this new methodology. The proposed method will allow design of truly homogenous assays. This methodology will be initially developed for proteins having natural sequence specific DNA binding activity. Subsequently, this methodology will be expanded to include the proteins which do not possess natural sequence specific nucleic acid binding activity. There will be two phases of this project. In the first phase we will use the model protein systems to provide "proof of principle" evidence that our approach can be developed for a DNA-binding protein and that it can be expanded to proteins lacking such activity. In the second phase of the project we will apply the concepts developed in the first phase of the project to generate "molecular beacons" which will recognize four cancer-related targets: p53 - a tumor suppressor whose inactivation is the most common defect in cancer cells, NF-kB - transcription factor involved in regulation of many genes and found to be constitutively active in many tumors, p16(INK4A) - a tumor suppressor involved in cell cycle regulation whose mutations have been found in greater than 70 different types of tumor cells, and p27(Kip1) - a cell-cycle inhibitor whose cellular levels were shown to be an important prognostic marker in breast cancer patients. We expect that the methodology we propose to develop will find broad applications in basic cancer research, molecular diagnosis of disease, identification of therapeutic markers and targets, and in characterization of response to pharmaceuticals.