A feasibility study of genetically encoded fluorescent gold nanoclusters

基因编码荧光金纳米团簇的可行性研究

• 批准号: 7492027
• 负责人: Jan T. Liphardt
• 金额: $ 24.46万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7492027
• 关键词: Address; Adverse effects; Anatomy; Animal Model; Biological Models; Biological Sciences; Biomedical Engineering; Biomedical Research; Biophysics; Cell physiology; Cells; Cellular Morphology; Chemical Engineering; Chimeric Proteins; Class; Collaborations; Collecting Cell; Complex; Cytoplasm; DNA biosynthesis; Data; Development; Disease Progression; Early Diagnosis; Electron Microscopy; Electrons; Embryo; Ensure; Feasibility Studies; Fishes; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Freezing; Functional disorder; Gene Delivery; Genetic; Gold; Growth; Hela Cells; Hereditary Disease; Image; In Vitro; Individual; International; Intracellular Transport; Ions; Label; Larva; Life; Localized; Measurement; Metals; Methodology; Modality; Molecular Machines; Molecular Toxicology; Monitor; Numbers; Optics; Oregon; Peptide Library; Peptides; Physics; Physiology; Plasmids; Positioning Attribute; Proteins; Public Health; Reporter Genes; Research; Research Personnel; Resolution; Ribosomes; Role; Signal Transduction; System; Technology; Testing; Time; Tissue Engineering; Tissues; Toxic effect; Toxicology; United States National Institutes of Health; Validation; Vertebrates; Water; Whole Organism; Work; X ray microscopy; Zebrafish; day; gold tetrachloride, acid; human disease; in vivo; light microscopy; mutant; nephrotoxicity; novel; research study; single molecule; size; tissue/cell culture; tool; transcription factor

DESCRIPTION (provided by applicant): Molecular machines are responsible for almost all central cellular functions and include the splicosome, RNA-induced interference complex, and ribosome. Biomedical researchers would like to follow as a function of time the position, composition, and structural and functional dynamics of single molecular machines inside living cells. Certain gold clusters are small, water soluble, brightly fluorescent, and photostable and have significant potential as labels for in vivo single molecule studies. However, gold clusters are not currently genetically encoded and it is not clear how to deliver them into the cytoplasm of living cells. Our hypothesis is that these two limitations can be resolved by specifically growing gold clusters inside living cells using scaffolded peptides. We will test this hypothesis with three specific research thrusts. 1. We will quantity the potential adverse effects of gold ions at the concentrations needed for intracellular cluster formation. HeLa tissue culture cells and zebrafish embryos/larvae will be exposed to chloroaurate for minutes to days. Potential adverse effects will be assessed by optical and electron microscopy. 2. We will quantify nonspecific labeling backgrounds and establish the ability of certain scaffolded peptides to direct in vivo cluster growth. In vivo cluster growth will be monitored with fluorescence and electron microscopy. 3. We will generate novel cluster nucleation peptides that perform well inside living cells. Scaffolded peptide libraries will be expressed in HeLa cells. Cells that become fluorescent upon gold exposure will be isolated by fluorescence-activated cell-sorting (FACS). Subsequent hit validation will involve epi-fluorescence, confocal, and electron microscopy. NIH PAR-07-234 has six aims; the proposed technology addresses PAR-07-234 objective 1, the development of new probes for light microscopy, objective 3, development of new classes of genetically encoded probes, and objective 4, genetically-encoded probes for electron and X-ray microscopy. As required by PAR-07-234, the proposed technology is unproven and novel, since 1) potential adverse effects have not been fully evaluated, especially not in a vertebrate model system, 2) it is not known whether fluorescent gold clusters can be specifically grown in vivo, 3) the ability of the technology to facilitate in vivo single molecule experiments and correlative optical, electron, and soft x-ray studies remains to be established, and 4) we will use FACS to identify scaffolded peptides that efficiently direct cluster growth in vivo. A multi-disciplinary team has been assembled to bring the needed expertise to these tasks. The three investigators are experts in single-molecule biophysics, optical and electron microscopy, development of new biomedical research tools, zebrafish physiology, and peptide-controlled synthesis of materials. They will be supported by two international experts in gene delivery, tissue engineering, and metal toxicology in zebrafish. The collaboration comprises researchers from Physics, Bioengineering, and Chemical Engineering (UC Berkeley), the Life Sciences and Physical Biosciences Depts. of Lawrence Berkeley Lab, and the Oregon State U. Dept. of Environmental and Molecular Toxicology. The proposed research will establish the feasibility of genetically encoded gold clusters. Such probes would represent a fundamentally new labeling technology differing from current methodologies by combining photostable fluorescence, genetic encoding, small size, and visibility in optical, electron, and X-ray microscopy ('multi-modality'). This research may greatly facilitate basic biomedical research by giving researchers a powerful new tool for watching how molecular machines work in their native context with high spatial and temporal resolution. Such measurements will reveal the roles of proteins within their tissue context and help to dissect molecular disease mechanisms, such as how mutant proteins disrupt interactions between transcription factors and impair the DNA replication machinery. The photostability of the probes may allow extended monitoring of individual proteins, enabling studies of gradual disease progression within cells and tissues. The brightness of the probes may permit early detection of cellular and tissue dysfunction in model systems. To ensure public health significance, much of the research will involve zebrafish, a vertebrate animal model system for an increasing number of human diseases.

描述（由申请人提供）：分子机器几乎负责所有中央细胞功能，其中包括接头，RNA诱导的干扰复合物和核糖体。生物医学研究人员希望随着时间的函数遵循活细胞内部分子机器的位置，组成以及结构和功能动力学。某些金簇很小，水溶性，明亮的荧光和光稳定性，并且具有在体内单分子研究的标签上具有巨大的潜力。但是，当前没有遗传编码的金簇，尚不清楚如何将它们输送到活细胞的细胞质中。我们的假设是，可以通过使用脚手架肽在活细胞内特异性种植金簇来解决这两个局限性。我们将通过三个特定的研究推力来检验这一假设。 1。我们将在细胞内簇形成所需的浓度下量化金离子的潜在不利影响。 HeLa组织培养细胞和斑马鱼胚胎/幼虫将暴露于氯龙几分钟到几天。潜在的不良反应将通过光学显微镜和电子显微镜进行评估。 2。我们将量化非特异性标记背景，并确定某些脚手架肽直接在体内簇生长的能力。体内簇生长将通过荧光和电子显微镜进行监测。 3。我们将生成在活细胞内表现良好的新型簇成核肽。脚手架肽库将在HeLa细胞中表达。荧光激活的细胞分类（FACS）将分离出荧光后荧光的细胞。随后的HIT验证将涉及荧光，共聚焦和电子显微镜。 NIH PAR-07-234有六个目标；提出的技术涉及PAR-07-234目标1，新的光学显微镜探针的开发，目标3，新的遗传编码探针的开发以及目标4，电子和X射线显微镜的遗传编码探针。 As required by PAR-07-234, the proposed technology is unproven and novel, since 1) potential adverse effects have not been fully evaluated, especially not in a vertebrate model system, 2) it is not known whether fluorescent gold clusters can be specifically grown in vivo, 3) the ability of the technology to facilitate in vivo single molecule experiments and correlative optical, electron, and soft x-ray studies remains to be established, and 4）我们将使用FACS识别有效直接在体内簇增长的脚手架肽。一支多学科团队已经组装，以将所需的专业知识带入这些任务。这三名研究人员是单分子生物物理学，光学和电子显微镜，新生物医学研究工具的开发，斑马鱼生理学以及材料控制肽控制的合成的专家。斑马鱼的基因递送，组织工程和金属毒理学方面的两名国际专家将支持它们。该合作包括物理，生物工程和化学工程（UC Berkeley），生命科学和物理生物科学部的研究人员。劳伦斯·伯克利实验室以及俄勒冈州环境和分子毒理学系拟议的研究将确定遗传编码的金簇的可行性。这些探针将通过结合光稳定荧光，遗传编码，小尺寸和光学，电子和X射线显微镜（“多模式”）中的光稳定荧光，遗传编码，小尺寸和可见性，从而代表与当前方法不同的新标记技术。这项研究可能会通过为研究人员提供一个有力的新工具来观察分子机器如何在其本地情况下以高空间和时间分辨率的方式工作，从而极大地促进了基本的生物医学研究。这种测量将揭示蛋白质在其组织环境中的作用，并有助于剖析分子疾病机制，例如突变蛋白如何破坏转录因子之间的相互作用和损害DNA复制机制。探针的光稳定性可能允许对单个蛋白质进行扩展监测，从而可以研究细胞和组织内的逐渐疾病进展。探针的亮度可能允许在模型系统中早期检测细胞和组织功能障碍。为了确保公共卫生的重要性，许多研究将涉及斑马鱼，斑马鱼是一种脊椎动物模型系统，用于越来越多的人类疾病。