Near-infrared fluorescent probes and optogenetic tools

摘要 近红外区哺乳动物组织的高光学透明度、低光散射和低自发荧光 红外光谱范围开辟了非侵入性成像，传感和细胞光调节的可能性 生物化学、组织代谢和生理学。由于近红外吸收和使用 作为发色团在所有哺乳动物组织中产生的血红素衍生胆绿素，细菌光敏色素是 目前，用于开发遗传编码的光学试剂的优选分子模板用于免疫荧光， 体内应用。在这里，我们提出了各种近红外成像探针和光遗传学工具。我们将 专注于开发具有更高亮度，更大的红移， 表现出光转化，以及寻找新的天然近红外吸收蛋白质作为初始 模板这些探针应导致更深和时空精确的体内成像。我们还将 专注于可视化和操纵细胞和整个生物体中的内源性分子， 近红外荧光蛋白与遗传编码的纳米抗体的抗原稳定融合物。这将 允许各种细胞内抗原的无背景检测和蛋白质的抗原依赖性调节 和细胞功能。此外，我们还将开发近红外光遗传学模块， 在哺乳动物细胞中的性能，并使用它们来设计光控酶，受体和细胞。这 应该开辟了用户定义的细胞和生物体功能在体内调节的可能性。所有 所提出的近红外探针将在可见光范围内对现有试剂进行光谱补充。计划 研究还将扩展我们对光敏色素的光化学和光诱导信号的基本知识 光感受器新的遗传编码荧光探针和光遗传学工具将推动这一发展 更灵敏的体内成像和光控技术，基因传递策略的优化， 开发有针对性的非侵入性照明，以及改进组织和动物中的光学读数。

ABSTRACT High optical transparency, low light-scattering and low autofluorescence of mammalian tissues in the near- infrared spectral range open up the possibility for non-invasive imaging, sensing and light-regulation of cellular biochemistry, tissue metabolism and physiology in mammals. Due to near-infrared absorption and the use of heme-derived biliverdin produced in all mammalian tissues as a chromophore, bacterial phytochromes are currently the preferred molecular templates for the development of genetically encoded optical reagents for in vivo applications. Here we propose a variety of near-infrared imaging probes and optogenetic tools. We will focus on the development of fluorescent proteins with higher brightness, greater bathochromic shift and exhibiting photoconversion, as well as on the search for new natural near-infrared absorbing proteins as initial templates. These probes should lead to deeper and spatiotemporally precise in vivo imaging. We will also focus on the visualization and manipulation of endogenous molecules in cells and whole organisms using antigen-stabilized fusions of near-infrared fluorescent proteins with genetically encoded nanobodies. This will allow background-free detection of various intracellular antigens and antigen-dependent regulation of protein and cellular functions. In addition, we will develop near-infrared optogenetic modules with improved performance in mammalian cells and use them to engineer light-controlled enzymes, receptors and cells. This should open up possibilities for user-defined regulation of cellular and organismal functions in vivo. All proposed near-infrared probes will spectrally complement existing reagents in the visible range. Planned studies will also expand our basic knowledge of photochemistry and light-induced signaling of phytochrome photoreceptors. Novel genetically encoded fluorescent probes and optogenetic tools will drive the development of more sensitive in vivo imaging and light-control technologies, optimization of gene delivery strategies, development of targeted non-invasive illumination, and refinement of optical readouts in tissues and animals.