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

摘要 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蛋白质印迹和酶联免疫吸附分析等技术。然而，开发特定蛋白质的分析方法是耗时的，并且是高通量筛选开发、生物标记物表征和许多基础科学研究项目的瓶颈。检测中的蛋白质 细胞同样困难，通常需要通过固定细胞和免疫荧光分析来牺牲细胞。目前，还没有允许在活细胞中实时监测内源性表达蛋白质水平的技术。该项目的目标是开发一种在体外和体内环境中检测蛋白质的通用和简单的方法。我们开发了一类新的基于寡核苷酸的 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。 该传感器的功能是在分析物结合时将原本非荧光的分子转化为荧光构型。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。我们描述了涉及在体内表达基因编码传感器的实验 检测细胞生长和分裂过程中的特定内源蛋白质。这种方法有可能比目前基于荧光共振能量转移(FRET)的基因可编码探针具有更广泛的用途。我们还描述了一种简单的方法来产生蛋白质传感微阵列 传感器。在这项技术中，生物样品将不需要使用荧光标记进行化学衍生化等样品处理。这种无标记的方法有可能为组织样本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在体外蛋白质定量中的应用。总之，本申请中描述的实验描述了一种多功能传感器技术，它将从根本上将新的和广泛有用的技术引入整个生物医学 研究社区。

ABSTRACT 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.