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.
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.
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