Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging

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使用单个荧光分子的显微成像技术的最新进展已经导致超分辨率信息，即，已经确定了细胞中物体的位置和形状，分辨率超过标准衍射极限。这些方法可以统称为单分子主动控制显微术（SMACM），因为单个发射分子被用作纳米级光源，并且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发射。光活化荧光蛋白融合已用于SMACM，但这些发射体很大，可能会扰乱生物系统。虽然一些发射体如量子点提供了高的光稳定性，但同时需要许多额外的性质用于细胞中的高级单分子成像，如易于功能化，控制光物理学和光化学，以及易于靶向特定的细胞结构。有机合成可以制造具有多个定制功能的大量“小”分子，并且本申请利用这种高度灵活性来开发具有主动控制能力的新的靶向单分子发射器。该研究将利用三个相互关联的推力来解决3-D超分辨率成像的问题，这三个推力联合收割机结合了有机合成、单分子成像、用于细胞靶向的化学和细菌细胞中的调节蛋白定位。首先，有机合成将产生具有“开启”能力的新荧光团，其中化学反应性仅在允许两个原荧光团反应时用于产生发射，或者其中二次光化学照明原位产生荧光分子。二次照明也将用于光电开关分子的打开和关闭，以进行额外的控制。开启概念的实用性在于，仅在需要的地方可以更容易地产生荧光;因此背景较低。第二个目标是将荧光标记选择性地定位到细胞中的蛋白质和RNA上。这将分别通过N-末端半胱氨酸标记和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特定的亚波长结构，无论是在真核细胞还是在微小的细菌细胞中。这项研究的结果将大大扩展高分辨率探针在单分子水平上用于细胞成像的可用性，从而使人们能够更深入地了解细胞功能。通过提供一个新的可控和有针对性的单分子发射器阵列，研究人员非侵入性地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，并且为了理解细胞的操作，光学成像由于其通常非侵入性的特性、其从远处评估的能力以及其观察时间依赖性动力学过程的能力而长期以来一直是一种基本方法。在细胞中，许多小分子机器一次运行一个，因此科学家们现在经常一个接一个地观察单个分子，以检查每个分子的行为，而不是平均许多不等价的副本。为了在几十nm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签，这项拟议的研究结合了四名研究人员的技能，设计，合成和优化了一种用于标记活细胞中单个蛋白质和RNA的大型新型分子。

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.