Actively Controlled and Targeted Single-Molecule Probes for Cellular Imaging

描述(申请人提供)：用于细胞成像的主动控制和靶向的单分子探针使用单荧光分子的显微成像技术的最新进展导致了超分辨率信息，即以超过标准衍射极限的分辨率确定细胞中对象的位置和形状。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几个分子发射。可光激活的荧光蛋白融合已经用于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，提供了很高的光稳定性，但先进的单分子细胞成像同时需要许多额外的性质，如易于功能化，光物理和光化学的控制，以及易于靶向特定的细胞结构。有机合成可以制造出大量具有多种定制功能的“小”分子，目前的应用是利用这种高度的灵活性来开发具有主动控制能力的新的、有针对性的单分子发射器。这项研究将通过三个相互连接的推力来解决三维超分辨率成像的问题，这三个推力结合了四名研究人员的技能，他们是有机合成、单分子成像、细胞靶向的化学和细菌细胞中调节蛋白定位的专家。首先，有机合成将产生具有“开启”能力的新的荧光团，即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。二次照明也将被用来开启和关闭分子的光开关，以实现额外的控制。开启概念的用处在于，只有在需要的地方才能更容易地产生荧光；因此背景较低。第二个推力涉及选择性地将荧光标记定位于细胞中的蛋白质和RNA。这将分别通过N-末端半胱氨酸标记和RNA适配子生成来完成。最后，为了验证和挑战荧光团的开发，将在单分子水平上使用新的发射器来成像真核细胞和微小细菌细胞中特定的亚波长结构。这项研究的结果将大大扩大在单分子水平上用于细胞成像的高分辨率探针的可用性，从而使人们能够更深入地了解细胞功能。通过提供大量可控和有针对性的单分子发射器阵列，研究人员非侵入性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区域。公共卫生相关性：对生物系统的了解与解开疾病机制密切相关，而为了了解细胞的运作，光学成像长期以来一直是一种基本的方法，因为它通常是非侵入性的，能够从远处进行评估，并且能够观察依赖时间的动态过程。在细胞中，许多小分子机器一次只运行一个，因此科学家们现在例行地观察单个分子，一个接一个地检查每个分子的行为，而不是平均计算许多不同的副本。为了在几十纳米的空间尺度上观察细胞中的单分子，需要新的主动可控和可靶向的发射标记，这项拟议的研究结合了四名研究人员的技能来设计、合成和优化一大类用于标记活细胞中单个蛋白质和RNA的新型分子。

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.