Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells.
Genetically encoded FRET sensors for visualizing metabolites with subcellular resolution in living cells.
复制标题
基因编码的 FRET 传感器，用于以亚细胞分辨率可视化活细胞中的代谢物。
DOI：
10.1104/pp.104.900151
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Frommer,WolfB
中科院分区：
文献类型：
--
作者：
Looger,LorenL;Lalonde,Sylvie;Frommer,WolfB
有什么工具可以确定生理环境中特定分子种类的浓度？是否有可能可视化其浓度在器官、组织或细胞中的变化？有没有一种方法可以检测代谢物水平在环境刺激下的变化？这些变化可以实时监控吗？多个分析物可以同时测量吗？这些测量可以对各种结构和功能分析物类进行吗？这些都是代谢组学这一新兴领域的核心问题。目前没有可用的技术以令人满意的方式解决这些问题。非水分馏法是静态的，侵入性的，没有细胞分辨率，对伪影很敏感。核磁共振成像和正电子发射断层扫描等光谱方法提供了动态数据，但空间分辨率较差。遗传编码分子传感器的发展，将目标分子与识别元件的相互作用转化为宏观可观察到的，通过一个或多个报告元件的变弹性调节，可能为一些问题提供答案。识别元件可以简单地结合靶标，结合并酶转化靶标，或者可以作为靶标的底物，如在蛋白酶传感器的构建中使用特定的靶标序列（Nagai和Miyawaki， 2004）。最常见的报告元件是绿色荧光蛋白（GFP； Fehr等人，2002）的立体分离的供体-受体荧光共振能量转移（FRET）对光谱变体，尽管单个荧光蛋白（Doi和Yanagawa， 1999）或酶（Guntas和Ostermeier， 2004）也是可行的。一些分子传感器另外使用构象驱动器（最常见的是与识别元件的一个构象状态结合的肽）来放大对报告元件的变构效应和由此产生的输出
What tools are there to determine the concentration of a particular molecular species in a physiological environment? Is it possible to visualize how its concentration varies across an organ, tissue, or cell? Is there a way to detect how metabolite levels change in response to environmental stimuli? Can these changes be monitored in real time? Can multiple analytes be measured simultaneously? Can these measurements be performed for a variety of structural and functional analyte classes? These are the central questions in the young field of metabolomics. No currently available technology addresses these issues in a satisfactory manner. Nonaqueous fractionation is static, invasive, has no cellular resolution, and is sensitive to artifacts. Spectroscopic methods such as nuclear magnetic resonance imaging and positron emission tomography provide dynamic data, but poor spatial resolution.The development of genetically encoded molecular sensors, which transduce an interaction of the target molecule with a recognition element into a macroscopic observable, via allosteric regulation of one or more reporter elements, may provide answers to some of the questions. The recognition element may simply bind the target, bind and enzymatically convert the target, or may serve as a substrate for the target, as in the use of a specific target sequence in the construction of a protease sensor (Nagai and Miyawaki, 2004). The most common reporter element is a sterically separated donor-acceptor fluorescence resonance energy transfer (FRET) pair of spectral variants of the green fluorescent protein (GFP; Fehr et al., 2002), although single fluorescent proteins (Doi and Yanagawa, 1999) or enzymes (Guntas and Ostermeier, 2004) are viable as well. Some molecular sensors additionally employ a conformational actuator (most commonly a peptide which binds to one conformational state of the recognition element) to magnify the allosteric effect upon and resulting output of the reporter element