Fluorescent Chemical Tags for Single-Molecule Imaging in Cells

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和特定的挑战主题06-GM-102*。化学家/生物学家合作促进工具开发。这项资助的目的是开发荧光化学标签，既具有高光子计数，又具有细胞渗透性和可溶性，以实现细胞中的单分子检测。单分子成像现在可以在体外进行，并为理解复杂生物组装的分子机制提供了突破，因为它允许直接观察单个复合物的动力学-但体内单分子成像仅由该领域的少数领先专家进行。在我们看来，常规细胞单分子成像的主要障碍是难以用合适的荧光团选择性地标记细胞中的蛋白质。荧光蛋白没有足够高的光子计数，无法在细胞中用单分子分辨率轻易检测到，而为体外生物物理学开发的具有高光子计数的有机荧光团在细胞中表现不佳。 公共卫生相关性：在这里，我们提出，化学标签，包括我们的商业TMP标签可以提供遗传编码和细胞中的单分子成像所需的有机荧光团标记的组合。我们组建了一个跨学科的团队，其中包括化学标签方面的化学生物学家专家（PI康沃尔语）、精通荧光显微镜的物理化学家（合作PI考夫曼）、研究剪接体反应机制的领先生物化学家（合作PI摩尔）和将最先进的显微镜应用于细胞运动的细胞生物学家（合作PI Sheetz），以实现这一目标。我们建议通过（1）挑战酵母细胞提取物中剪接体单分子成像的化学标签，（2）开发适用于细胞中单分子成像的荧光化学标签，以及（3）开发具有高分辨率成像专用特性的化学标签来实现这一目标。

DESCRIPTION (Supplied by the Applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic 06-GM-102* .Chemist/biologist collaborations facilitating tool development. The objective of this grant is to develop fluorescent chemical tags that both have high photon counts and are cell permeable and soluble to enable single-molecule detection in cells. Single-molecule imaging can now be carried out in vitro and has provided a breakthrough for understanding the molecular mechanisms of complex biological assemblies because it allows the dynamics of individual complexes to be directly observed--but in vivo single-molecule imaging has only been carried out by a handful of leading experts in the field. In our opinion a major barrier to routine single-molecule imaging in cells is the difficulty of labeling proteins selectively in cells with suitable fluorophores. The fluorescent proteins do not have high enough photon counts to be readily detected with single molecule resolution in cells, and the organic fluorophores with high photon counts developed for in vitro biophysics do not behave well in the cell. PUBLIC HEALTH RELEVANCE: Here, we propose that chemical tags including our commercial TMP-tag can provide the combination of genetic encoding and an organic fluorophore label needed for single-molecule imaging in cells. We have assembled an interdisciplinary team of a chemical biologist expert in chemical tags (PI Cornish), a physical chemist skilled in fluorescence microscopy (co-PI Kaufman), a leading biochemist studying the mechanism of the spliceosome reaction (co-PI Moore), and a cell biologist who is a pioneer in the application of state-of-the-art microscopy to cell motility (co-PI Sheetz) to meet this objective. We propose to meet this objective by (1) challenging the chemical tags for single-molecule imaging of the spliceosome in yeast cell extracts, (2) developing fluorescent chemical tags suitable for single-molecule imaging in cells, and (3) developing chemical tags with specialized properties for high-resolution imaging.