Near-Infrared Fluorescent Proteins, Biosensors and Optogenetic Tools

摘要 近红外光由于血红蛋白的低吸光度而有利于在哺乳动物组织中成像， 黑色素和水。因此，用于最佳成像的荧光蛋白质、生物传感器和光遗传结构， 哺乳动物的光学读数和光操作应该在 近红外窗。有趣的是，天然细菌光敏色素利用低分子胆绿素， 在大多数哺乳动物组织中发现，作为一种光反应发色团。由于它们的近红外吸收 细菌光敏色素是设计光学分子工具的首选模板 哺乳动物。基于对细菌光致变色剂的光化学和结构的分析，我们提出了一种 各种可能的细菌光敏色素荧光蛋白、生物传感器和光遗传工具。这个 提出了此类探头的设计策略和实验考虑。近红外荧光 蛋白质和生物传感器将把为传统显微镜开发的方法扩展到深部组织 活体宏观包括多色细胞和组织标记、荧光共振能量转移、细胞 光激活和跟踪，以及检测组织中的酶活性和代谢物。近红外线 光遗传工具将允许直接对活动物的生物化学和生理进行非侵入性的光控制 穿透皮肤。此外，所有以细菌光敏色素为基础的试剂将在光谱上补充现有的 在可见光范围内用基因编码的探测器。计划中的探测还将扩大我们对 自然界中光敏色素的光化学和光诱导信号传递。近红外探头的可用性 将进一步刺激新的活体成像和光操纵技术的发展，优化 向体内特定细胞和组织传递基因的策略，靶向非侵入性照明的设计， 以及改进光学读数。总体而言，这将导致广泛的化学和非侵入性研究 代谢状态，以及完整组织和整个活着的哺乳动物中的分子和细胞相互作用。

ABSTRACT Near-infrared light is favorable for imaging in mammalian tissues due to low absorbance of hemoglobin, melanin and water. Therefore, fluorescent proteins, biosensors and optogenetic constructs for optimal imaging, optical readout and light manipulation in mammals should have fluorescence and action spectra within the near-infrared window. Interestingly, natural bacterial phytochromes utilize the low molecular weight biliverdin, found in most mammalian tissues, as a photoreactive chromophore. Due to their near-infrared absorbance bacterial phytochromes are preferred templates for designing optical molecular tools for applications in mammals. Based on the analysis of the photochemistry and structure of bacterial phytochromes we suggest a variety of possible bacterial phytochrome-based fluorescent proteins, biosensors, and optogenetic tools. The design strategies and experimental considerations for such probes are proposed. Near-infrared fluorescent proteins and biosensors will extend the methods developed for conventional microscopy into a deep-tissue in vivo macroscopy including multicolor cell and tissue labeling, fluorescence resonance energy transfer, cell photoactivation and tracking, and detection of enzymatic activities and metabolites in tissues. The near-infrared optogenetic tools will allow noninvasive light-control of biochemistry and physiology of a living animal directly through the skin. Moreover, all bacterial phytochromes-based reagents will spectrally complement existing genetically encoded probes in the visible range. The planned probes also will expand our basic knowledge of photochemistry and light-induced signaling of phytochromes in nature. Availability of the near-infrared probes will further stimulate the development of novel in vivo imaging and light-manipulation technologies, optimization of strategies for gene delivery to specific cells and tissues in vivo, design of targeted noninvasive illumination, and refining optical readouts. Overall, this will result in a wide range of noninvasive studies of chemical and metabolic status, as well as molecular and cellular interactions in intact tissues and whole living mammals.