Near-infrared fluorescent probes and optogenetic tools

摘要 近红外波段哺乳动物组织的高光学透明度、低光散射和低自发荧光 红外光谱范围为细胞的非侵入性成像、传感和光调节提供了可能 哺乳动物的生物化学、组织代谢和生理学。由于近红外吸收和使用 血红素衍生的胆绿素在所有哺乳动物组织中作为发色团产生，细菌光敏色素是 目前用于开发基因编码的光学试剂的首选分子模板 活体应用。在这里，我们提出了各种近红外成像探针和光遗传工具。我们会 重点开发亮度更高、红移更大的荧光蛋白和 展示了光转化，以及作为初始的寻找新的天然近红外吸收蛋白 模板。这些探头应该会带来更深层次和时空上精确的活体成像。我们还将 专注于细胞和整个生物体中内源分子的可视化和操纵 抗原稳定的近红外荧光蛋白与基因编码的纳米体的融合。这将是 允许无背景检测各种细胞内抗原和对蛋白质的抗原依赖性调节 和细胞功能。此外，我们还将开发经过改进的近红外光伏组件 在哺乳动物细胞中发挥作用，并利用它们来设计光控酶、受体和细胞。这 应该为在体内对细胞和组织功能进行用户定义的调节打开可能性。全 拟议的近红外探测器将在可见光范围内对现有试剂进行光谱补充。计划中的 研究还将扩展我们关于光化学和光诱导的光敏色素信号的基本知识 光感受器。新的基因编码的荧光探针和光遗传工具将推动这一发展 更灵敏的活体成像和光控制技术，基因传递策略的优化， 开发有针对性的非侵入性照明，并改进组织和动物的光学读数。

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.