MOLECULAR BEACONS FOR PROTEIN DETECTION

描述（由申请人提供）：以高度多路复用方式快速检测和定量特定蛋白质的能力对于基础科学研究、药物研究和生物医学研究具有重要意义。 筛查应用和临床诊断。在本申请中，我们提出开发一种新的方法，该方法将允许产生能够同时识别特定蛋白质分子并发出识别特定蛋白质分子的信号的报告分子。这些“分子信标”产生的信号将很容易测量荧光强度， 促进这种新方法的高通量使用。所提出的方法将允许设计真正同质的测定。该方法最初将开发用于具有天然序列特异性DNA结合活性的蛋白质。随后，该方法将扩展到包括不具有天然序列特异性核酸结合活性的蛋白质。该项目将分为两个阶段。在第一阶段，我们将使用模型蛋白质系统提供“原理证明”证据，证明我们的方法可以开发用于DNA结合蛋白，并且可以扩展到缺乏这种活性的蛋白质。在项目的第二阶段，我们将应用项目第一阶段开发的概念， 产生“分子信标”，它将识别四个癌症相关的目标：p53 -肿瘤抑制因子，其失活是癌细胞中最常见的缺陷，NF-κ B-转录因子参与许多基因的调节，并发现在许多肿瘤中具有组成性活性，p16（INK 4A）-肿瘤抑制因子，其突变涉及细胞周期调节， 在超过70种不同类型的肿瘤细胞中发现，以及p27（Kip 1）-细胞周期抑制剂，其细胞水平被证明是乳腺癌患者的重要预后标志物。我们希望我们提出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，以及对药物反应的表征。

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.