Novel fluorescent sensors for simple, sensitive, and specific protein detection

描述（由申请人提供）：蛋白质的检测基本上是所有生物医学研究的基础。目前检测蛋白质的策略包括Western blotting和酶联免疫吸附测定等技术。然而，开发特定蛋白质的检测方法是耗时的，并且是高通量筛选开发、生物标志物表征和许多基础科学研究项目的瓶颈。检测细胞中的蛋白质同样困难，通常需要牺牲细胞，然后进行免疫荧光测定。目前，还没有技术允许在活细胞中实时监测内源性表达蛋白的水平。这个项目的目标是开发一种通用的和简单的方法来检测蛋白质在体外和体内的设置。我们已经开发了一种新型的基于寡核苷酸的传感器，并且我们已经证明这些传感器允许对特定分析物进行荧光检测。传感器的功能是在分析物结合时将非荧光分子转化为荧光结构。我们建议开发一种可推广的方法，允许为几乎任何蛋白质开发传感器。我们描述了在细胞生长和分裂过程中表达基因编码传感器以检测特定内源性蛋白质的实验。这种方法具有比当前基于荧光共振能量转移（FRET）的基因可编码探针更通用的潜力。我们还描述了一种使用这些传感器生成蛋白质传感微阵列的简单方法。在这种技术中，生物样品将不需要样品处理，如荧光标记的化学衍生化。这种无标记的方法有可能为组织样品的蛋白质组学分析提供一种简单的方法。我们还描述了这些传感器用于体外蛋白质定量的使用。总之，本应用程序中描述的实验描述了一种多功能传感器技术，它将为整个生物医学研究界引入基本新颖且广泛有用的技术。

DESCRIPTION (provided by applicant): The detection of proteins is fundamental to essentially all biomedical research. Current strategies to detect proteins include techniques such as Western blotting and enzyme-linked immunosorbent assays. However, developing assays for specific proteins is time consuming and a bottleneck in high throughput screen development, biomarker characterization, and many basic science research projects. Detecting proteins in cells is similarly difficult, and typically requires sacrificing the cell by fixation followed by immunofluorescence assays. Currently, there are no techniques that permit the levels of endogenously expressed proteins to be monitored in real time in living cells. The goal of this project is to develop a generalizable and simple method to detect proteins in an in vitro and in vivo setting. We have developed a novel class of oligonucleotide-based sensors, and we have demonstrated that these sensors permit the fluorometric detection of specific analytes. The sensor functions by converting an otherwise nonfluorescent molecule into a fluorescent configuration upon analyte binding. We propose to develop a generalizable method that would allow sensors to be developed for virtually any protein. We describe experiments that involve expressing genetically encoded sensors within cells to detect specific endogenous proteins during cell growth and division. This approach has the potential to be vastly more versatile that current fluorescence resonance energy transfer (FRET)-based genetically encodable probes. We also describe a simple approach to generate protein sensing microarrays using these sensors. In this technique, biological samples will not require sample processing such as chemical derivatization with a fluorescent tag. This label-free approach has the potential to provide a simple methodology for proteomic analyses of tissue samples. We also describe the use of these sensors for in vitro protein quantification. Together, the experiments described in this application describe a versatile sensor technology that will introduce fundamentally novel and widely useful technologies to the entire biomedical research community. PUBLIC HEALTH RELEVANCE: Detecting proteins is one of the most fundamental requirements in biomedical research. However, endogenous proteins cannot be detected in living cells in real time. Furthermore, simple, reagentless techniques for protein quantification in biological samples are not available. We have developed novel oligonucleotide-based sensors that become fluorescent upon exposure to specific analytes. This application describes a simple, sensitive, and specific method for using these sensors for detecting and quantifying proteins. This application also describes how these sensors can be used to monitor protein expression in cells in real time, and describes a novel protein-sensing microarray technology. These experiments will result in powerful new tools for protein detection that will markedly enhance and expand biomedical research.