Imaging single proteins in vivo with quantum dots

使用量子点对体内单个蛋白质进行成像

• 批准号: 7104026
• 负责人: SANFORD M SIMON
• 金额: $ 6.52万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7104026
• 关键词: Xenopus oocyte; bioimaging /biomedical imaging; exocytosis; fluorescence microscopy; membrane proteins; method development; nanotechnology; nuclear membrane; protein localization; protein structure function; protein transport

DESCRIPTION (provided by applicant): The long-term goals of this research are to develop robust methods for tracking single proteins in living cells. Recently developed technologies of protein splicing will be used to ligate fluorescent nanocrystals [quantum dot (QD)] derivatives to select proteins in living cells. The availability of such cellular reagents, in combination with modem fluorescence microscopy methods such as to total-internal-reflection microscopy and spectral imaging, will allow insight on protein activity that would be difficult to obtain using macromolecular measurements where protein activities are averaged. There are three specific aims in the proposal: Aim 1: To extend and optimize recently developed in vivo protein trans-splicing and expressed protein ligation approaches to allow the ligation of suitable QD derivatives to either cytosolic and integral membrane proteins. In addition, we will develop a conditional protein trans-splicing approach that will allow probes such as quantum dots to be ligated to proteins following a designated functional interaction. This will allow the cellular fate of "activated" proteins to be monitored. Aim 2: To develop methods for preparing monvalent QDs capable of being ligated to a single copy of a target protein in cells. In addition, to develop a strategy, based on fluorescence quenching, that will allow the fluorescence properties of a QD to be linked to the in vivo trans-splicing reaction. This will allow the fluorescence of a protein-QD ligation product to be distinguishable from unreacted QDs. Aim 3: To apply in vivo protein ligation and quantum dots to single proteins in vivo. The technology will be established in the context of two systems of biological interest: exocytosis and transport through the nuclear pore. For each system there is a set of questions that require the tracking of individual proteins. For example, exploring whether protein movement through the pore is primarily diffusive, driven by thermal fluctuations, or deterministic, driven by a molecular motor. Longer-term strategic directions will include trying to develop the technology for chemically tagging interacting proteins and technologies for tracking proteins for longer time periods as they move in three dimensions.

描述（由申请人提供）：这项研究的长期目标是开发用于跟踪活细胞中单蛋白的强大方法。 最近开发的蛋白质剪接技术将用于结合荧光纳米晶[量子点（QD）]衍生物，以选择活细胞中的蛋白质。 这种细胞试剂的可用性以及调制解调器荧光显微镜方法（例如总内内反射显微镜和光谱成像）将允许对蛋白质活性进行深入了解，而蛋白质活性很难使用蛋白质活性来获得平均蛋白质活性。 该提案中有三​​个具体目标： 目标1：扩展和优化最近开发的体内蛋白质移植和表达的蛋白结扎方法，以允许将合适的QD衍生物连接到胞质和整体膜蛋白上。 此外，我们将开发一种有条件的蛋白质移植方法，该方法将允许在指定的功能相互作用后将量子点（例如量子点）连接到蛋白质。 这将允许监测“激活”蛋白质的细胞命运。 AIM 2：开发方法来制备能够与细胞中靶蛋白的单个副本相连的质量QD。 此外，要根据荧光淬火制定策略，将允许将QD的荧光性能与体内移媒体反应相关。 这将使蛋白质QD连接产物的荧光与未反应的QD区分开。 目标3：将体内蛋白结扎和量子点应用于体内的单蛋白。 该技术将在两个生物学感兴趣系统的背景下建立：胞吞作用和通过核孔运输。 对于每个系统，都有一组需要跟踪各个蛋白质的问题。 例如，探索蛋白质通过孔的运动是否主要是扩散的，是由热波动驱动的还是由分子运动驱动的确定性。 长期战略方向将包括尝试开发用于化学标记相互作用蛋白质和技术以在三个维度移动时跟踪蛋白质的技术。