Molecular Fluorescent Toolkit

描述(申请人提供)：维多利亚水母的绿色荧光蛋白(GFP)和珊瑚的同源荧光蛋白(FP)已成为细胞成像的宝贵工具。与GFP变体不同的是，安东萨FP具有不同的颜色和功能，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。几个珊瑚虫FP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。最近发展了两种超分辨荧光技术：受激发射耗尽(STED)荧光显微镜和光活化定位显微镜(Palm)。随着双光子激光技术的发展，活体动物的深部组织活体成像技术已经得到了广泛的应用。然而，适用于这些成像技术的增强型单体FP只有两种颜色。我们对珊瑚虫FPS中生色团形成机制的分析表明，确实可以设计出具有新颖光谱和光化学特征的荧光探针。在现有单体FP的基础上，我们计划开发三种新的蛋白质标记，以补充现有的绿色和红色探针。其中包括单体可光激活FP(PA-FP)，它最初是暗的，但在紫光照射下在蓝色、橙色或远红区变得荧光(目标1)；具有大斯托克斯位移(LSS)发射的单体FP(LSSFP)，它在青色中吸收，但在橙色或远红色区域发光，我们进一步计划将其转化为可光激活的LSS-FP(目标2)；以及增强型单体远红FP，其亮度得到提高，并进一步向远红光移动，用于使用红色激光进行有效激发(目标3)。我们将应用定向分子进化技术，包括合理的基于结构的设计和候选蛋白质的随机突变，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，以分别优化用于活体成像的LSS-FP和用于Palm的PA-FP的光物理性质。我们将把突变过程与光谱和光化学变化联系起来，以深入了解负责荧光性质的发色团结构的分子进化，并将这些应用于下一轮分子进化。使用传统的荧光显微镜和超分辨率成像技术，将在体外彻底表征荧光变体，并将其作为活细胞中的融合标签。这项拟议研究的预期最终结果是一系列具有新荧光颜色的分子荧光工具，它们将与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，以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。增强型FRFP和新的LSSFP可用单波长双光子激光激发，将进一步推动深部组织和多色活体成像方法的发展。

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.