Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging

用于细胞成像的主动控制和靶向单分子探针

• 批准号: 7904851
• 负责人: William E Moerner
• 金额: $ 67.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904851
• 关键词: 3-Dimensional; Achievement; Aptamer Technology; Area; Bacteria; Behavior; Caulobacter crescentus; Cell physiology; Cells; Cellular Structures; Chemicals; Chemistry; Chimeric Proteins; Cilia; Collaborations; Complex; Coupled; Cysteine; Detection; Development; Disease; Ensure; Eukaryotic Cell; Fluorescence; Generations; Growth; Image; Imaging Techniques; In Situ; Individual; Label; Laboratories; Lead; Life; Light; Lighting; Location; Methods; Microscopic; Microscopy; Molecular Machines; N-terminal; Organic Synthesis; Pattern; Photochemistry; Positioning Attribute; Process; Property; Proteins; Quantum Dots; RNA; Research; Research Personnel; Resolution; Scheme; Scientist; Signaling Protein; Source; Structure; Synthesis Chemistry; System; Techniques; Testing; Time; active control; aptamer; base; biological systems; cellular imaging; cellular targeting; design; flexibility; fluorescence imaging; fluorophore; genetic regulatory protein; human SMO protein; in vivo; interest; multicatalytic endopeptidase complex; multidisciplinary; nanoscale; novel; object shape; operation; optical imaging; photoactivation; protein complex; public health relevance; single molecule; skills; small molecule; success

DESCRIPTION (provided by applicant): Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging Recent advances in microscopic imaging techniques with single fluorescent molecules have led to superresolution information, that is, the locations and shapes of objects in cells have been determined with resolution beyond the standard diffraction limit. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), because single emitting molecules are used as nanometer-scale light sources, and these emitters must be actively turned on and off to be sure that only a few molecules are emitting at any given time. Photoactivatable fluorescent protein fusions have been used for SMACM, but these emitters are large and may perturb the biological system. Though some emitters such as quantum dots provide high photostability, many additional properties are simultaneously required for advanced single-molecule imaging in cells, such as ease of functionalization, control of photophysics and photochemistry, and ease of targeting to specific cellular structures. Organic synthesis can make a huge array of "small" molecules with multiple tailored functionalities, and the present application makes use of this high degree of flexibility to develop new, targeted single-molecule emitters with active control capabilities This research will attack the problem of 3-D superresolution imaging with three interconnected thrusts which combine the skills of four investigators expert in organic synthesis, single-molecule imaging, chemistry for cellular targeting, and regulatory protein localization in bacterial cells. First, organic synthesis will generate new fluorophores with "turn-on" capability, where chemical reactivity is used to generate emission only when two protofluorophores are allowed to react, or where secondary photochemical illumination creates a fluorescent molecule in situ. Secondary illumination will also be used to photoswitch molecules on and off for additional control. The utility of the turn-on concept is that fluorescence can more easily be generated only where needed; hence backgrounds are lower. The second thrust involves selective targeting of the fluorescent labels to proteins and RNA in the cell. This will be accomplished by N-terminal cysteine labeling and RNA aptamer generation, respectively. Finally, to validate and challenge the fluorophore development, the new emitters will be used at the single-molecule level to image specific subwavelength structures, both in eukaryotic and in tiny bacterial cells. The results of this research will be to greatly extend the availability of high-resolution probes for cellular imaging at the single-molecule level, thus enabling a much deeper understanding of cellular functions. By providing a large new array of controllable and targeted single-molecule emitters, the ability of the researcher to noninvasively look inside cells will be extended into the nanoscale regime of the single-molecule emitters themselves. Public Health Relevance: The understanding of biological systems is intimately connected with unraveling disease mechanisms, and to understand the operation of the cell, optical imaging has long been an essential method by virtue of its generally noninvasive character, its capacity to assess from a distance, and its ability to observe time- dependent dynamical processes. In the cell, many small molecular machines operate one at a time, therefore scientists are now routinely observing individual single molecules, one by one, to examine the behavior of each without averaging over many inequivalent copies. To observe single molecules in cells at the spatial scale of a few tens of nm, new actively controllable and targetable emitting labels are required, and this proposed research combines the skills of four investigators to design, synthesize, and optimize a large and novel class of molecules for labeling individual proteins and RNA in living cells.

描述（由申请人提供）：用于细胞成像的主动控制和靶向单分子探针。单荧光分子显微成像技术的最新进展导致了超分辨率信息的出现，即细胞中物体的位置和形状已经以超过标准衍射极限的分辨率确定。这些方法可以统称为单分子主动控制显微镜（SMACM），因为单发射分子被用作纳米级光源，这些发射器必须主动打开和关闭，以确保在任何给定时间只有少数分子在发射。光激活荧光蛋白融合物已用于SMACM，但这些发射器很大，可能会扰乱生物系统。虽然量子点等一些发射体提供了高光稳定性，但在细胞中进行高级单分子成像同时需要许多额外的特性，例如易于功能化，光物理和光化学控制，以及易于靶向特定细胞结构。有机合成可以制造大量具有多种定制功能的“小”分子，目前的应用利用这种高度的灵活性来开发具有主动控制能力的新型靶向单分子发射器。本研究将通过三个相互关联的推力来解决三维超分辨率成像问题，这三个推力结合了四位研究人员在有机合成、单分子成像、细胞靶向化学、以及调节蛋白在细菌细胞中的定位。首先，有机合成将产生具有“开启”能力的新荧光团，只有当两个原荧光团被允许反应时，化学反应才能产生发射，或者二次光化学照明在原位产生荧光分子。二次照明也将用于光开关分子的打开和关闭，以进行额外的控制。开启概念的效用在于，荧光可以更容易地只在需要的地方产生；因此背景较低。第二个推力涉及到荧光标记对细胞中蛋白质和RNA的选择性靶向。这将分别通过n端半胱氨酸标记和RNA适体生成来完成。最后，为了验证和挑战荧光团的发展，新的发射器将在单分子水平上用于成像真核生物和微小细菌细胞中的特定亚波长结构。这项研究的结果将极大地扩展用于单分子水平细胞成像的高分辨率探针的可用性，从而能够更深入地了解细胞功能。通过提供大量新的可控和靶向单分子发射器，研究人员无创观察细胞内部的能力将扩展到单分子发射器本身的纳米级范围。公共卫生相关性：对生物系统的理解与揭示疾病机制密切相关，为了理解细胞的运作，光学成像一直是一种重要的方法，因为光学成像具有一般的非侵入性、远距离评估的能力和观察时间依赖性动态过程的能力。在细胞中，许多小分子机器一次只运行一个，因此科学家们现在通常是一个接一个地观察单个分子，以检查每个分子的行为，而不需要对许多不相等的复制进行平均。为了在几十纳米的空间尺度上观察细胞中的单个分子，需要新的主动可控和可靶向的发射标记，本研究结合四位研究者的技能，设计、合成和优化了一类大型的新型分子，用于标记活细胞中的单个蛋白质和RNA。