Molecular Fluorescent Toolkit

描述（由申请人提供）：来自维多利亚Aequorea水母的绿色荧光蛋白（GFP）和来自珊瑚虫珊瑚的同源荧光蛋白（FPs）已经成为细胞成像的宝贵工具。珊瑚虫FPs具有不同于GFP变体的颜色和特征，因此为分子标记和细胞内检测的新探针提供了强大的模板。一些珊瑚虫FPs已经被开发成生物技术工具。然而，光学显微镜方法和荧光成像方法的不断进步要求探针具有新的颜色和光化学性质。两种超分辨率荧光技术，受激发射耗尽（STED）荧光显微镜和光激活定位显微镜（PALM）是近年来发展起来的。随着双光子激光技术的进步，活体动物深部组织活体成像技术已经广泛应用。然而，适合这些成像技术的增强单体FPs仅存在两种颜色。我们对珊瑚虫FPs中发色团形成机制的分析表明，具有新的光谱和光化学特征的荧光探针确实可以被设计出来。在现有单体FPs的基础上，我们计划开发三种新的蛋白质标签，以补充现有的绿色和红色探针。这些包括单体光活化FPs (PA-FPs)，它们最初是深色的，但在紫光照射下在蓝色、橙色或远红色区域变成荧光（Aim 1）；具有大斯托克斯位移（LSS）发射（LSSFPs）的单体FPs，吸收青色，但在橙色或远红色区域发出荧光，我们进一步计划将其转化为光活化的LSS-FPs (Aim 2)；增强的单体远红色FP具有更高的亮度，并进一步向远红色转移，从而使用红色激光器进行有效激发（Aim 3）。我们将应用定向分子进化技术，包括基于合理结构的设计和候选蛋白的随机诱变，然后是流式细胞术和多孔板筛选。此外，将开发利用双光子激发和单分子表征的筛选方法，分别优化用于活体成像的LSS-FPs和用于PALM的PA-FPs的光物理性质。我们将把诱变过程与光谱和光化学变化联系起来，以便深入了解负责荧光特性的发色团结构的分子进化，并将这些应用于下一轮的分子进化。荧光变体将在体外和活细胞中的融合标签中进行彻底的表征，使用传统的荧光显微镜，以及超分辨率成像技术。这项研究的预期最终结果是一组具有新荧光颜色的分子荧光工具，这些荧光工具将像各自的绿色和红色蛋白质一样通用。由此产生的探针将扩展PA-FP技术，允许同时对几种细胞内蛋白质的定位和动态进行衍射限制或超分辨率PALM成像。增强的FRFP和新的lssfp，可以用单波长双光子激光激发，将进一步推进深部组织和多色活体成像方法。

DESCRIPTION (provided by applicant): The green fluorescent protein (GFP) from jellyfish Aequorea victoria and homologues fluorescent proteins (FPs) from Anthozoa corals have become invaluable tools for cell imaging. Anthozoa FPs is available in colors and with features unlike those of GFP variants and, thus, provides powerful templates for new probes for molecular labeling and intracellular detection. Several Anthozoa FPs have been already developed into biotechnological tools. Nevertheless, the continuing progress in optical microscopy methods and fluorescence imaging approaches requires probes with new colors and photochemical properties. Two super-resolution fluorescence techniques, stimulated emission depletion (STED) fluorescence microscopy and photoactivated localization microscopy (PALM), have been recently developed. With the improvement of two-photon lasers, a deep-tissue intravital imaging in live animals has become widely available. However, enhanced monomeric FPs suitable for these imaging techniques exist in two colors only. Our analysis of the chromophore formation mechanisms in Anthozoa FPs suggest that fluorescent probes with novel spectral and photochemical features can be indeed designed. On the basis of existing monomeric FPs we plan to develop three new types of protein labels complementary to the available green and red probes. These include monomeric photoactivatable FPs (PA-FPs), which are initially dark but become fluorescent in Blue, Orange or Far-red regions upon irradiation with violet light (Aim 1); monomeric FPs with large Stokes shift (LSS) emission (LSSFPs), which absorb in cyan but fluoresce in Orange or Far-Red regions, and which we further plan to convert into photoactivatable LSS-FPs (Aim 2); and an enhanced monomeric Far-Red FP with improved brightness and further shifted towards far-red for efficient excitation using red lasers (Aim 3). We will apply directed molecular evolution techniques consisting of rational structure-based design and random mutagenesis of candidate proteins, followed by flow cytometry and multiwell plate screening. Moreover, screening methods utilizing two-photon excitation and single-molecule characterization will be developed to optimize photophysical properties of LSS-FPs for intravital imaging and of PA-FPs for PALM, respectively. We will correlate the mutagenesis process with spectral and photochemical changes, in order to gain insight into the molecular evolution of chromophore structures responsible for fluorescence properties and will apply these to the next rounds of molecular evolution. The fluorescent variants will be thoroughly characterized in vitro and as fusion tags in live cells, using a conventional fluorescence microscopy, as well as the super-resolution imaging techniques. The anticipated end result of the proposed research is a collection of molecular fluorescent tools with new fluorescent colors that will be as versatile as the respective green and red proteins. The resulting probes will expand the PA-FP technology to allow diffraction-limited or super-resolution PALM imaging of localization and dynamics of several intracellular proteins simultaneously. The enhanced FRFP and new LSSFPs, excitable with a single wavelength two-photon laser, will further advance the deep-tissue and multicolor intravital imaging approaches.