Engineering bacterial phytochromes for near-infrared imaging in mammals

描述(由申请人提供)：对深层组织发育、代谢和致病过程的非侵入性监测将推动现代生物学的发展。在近红外(NIRW：650-900 nm)透明窗口(NIRW：650-900 nm)内，使用荧光探针对活体哺乳动物进行成像更可行，在该窗口中，血红蛋白和黑色素的吸光度显著降低，水的吸光度仍然很低。基因编码的探针中的生色团既可以由氨基酸自动催化形成，就像绿色荧光蛋白(GFP)类蛋白一样，也可以与脱辅基蛋白结合。在类绿色荧光蛋白家族中，大多数红移的荧光蛋白(FP)的激发和发射光谱完全或部分在NIRW之外，并且具有低亮度和适度的光稳定性。天然细菌光敏色素感受器利用低分子胆绿素作为发色团。结合胆绿素的BphPs与其他含发色团的蛋白质相比具有许多优点。首先，与非细菌光敏色素的发色团不同，胆绿素在哺乳动物中普遍存在。这使得BphP在哺乳动物细胞、组织和整个哺乳动物中的应用与传统的类似GFP的FP一样容易，而不需要通过外部溶液提供发色团。其次，BphP表现出近红外吸收和荧光，相对于任何其他光敏色素都是红移的，位于NIRW范围内。这使得BphP在光谱上补充了其他现有的生物光子学工具，如所有类似GFP的FFP和可用的光遗传工具。第三，独立的结构域结构和胆绿素光异构化引起的显著构象变化使BphPs成为设计各种光活化NIRFPs的模板。基于我们对BphP的光化学和结构变化的分析，我们计划开发三种新型的基于BphP的NIRFP。这些包括三种明亮的、光谱可分辨的近红外RFPs，可能称为短、中和长近红外RFPs(目标1)；可用非光毒性近红外光PA-NIRFPs光激活，这些PA-NIR RFPs最初是暗的，但在短、中或长-近红外光谱区变得荧光，以及这些近红外区域之间不可逆地(PS-NIRFPs)或重复(RS-NIRFPs)(目标2)；以及基于利用NIRFPs单体形式的可逆双分子荧光互补方法的蛋白质相互作用和磷酸化的近红外记者(目标3)。我们将应用基于合理结构的设计和候选蛋白质的随机突变的定向分子进化方法，然后是流式细胞术细菌细胞分选，在培养皿上筛选菌落，以及多孔板蛋白质鉴定。这些常规技术将允许筛选标准FP属性，如激发和发射波长、总体亮度、光稳定性、pH稳定性和在生理温度下的折叠。将开发新的高通量筛选方法，专门优化基于BphP的NIRFPs。选择高量子产率的NIRFP是BphP衍生FP的关键参数，将使用同时测量数千个菌落的时间分辨荧光寿命来执行。为了筛选与胆绿素的高亲和力，而胆绿素不能穿透细菌内膜，将采用脉冲追逐血红素加氧酶共表达产生胆绿素的方法，并将BphP NIRFPs靶向外源胆绿素可进入的细菌周质空间。有希望的NIRFP候选基因将直接在哺乳动物细胞中使用穿梭载体进行筛选，以优化哺乳动物细胞中的蛋白质折叠和稳定性，与内源性胆绿素的亲和力，并降低细胞毒性。优化的近红外探测器将在小鼠肿瘤模型中进行测试，并应用于活体哺乳动物的研究。由此产生的近红外探针将把荧光成像方法扩展到体内深部组织的宏观观察，包括多色细胞和组织标记、细胞光激活和跟踪、检测哺乳动物组织和整个动物的酶活性和蛋白质相互作用。

DESCRIPTION (provided by applicant): Non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biology. Imaging of live mammals using fluorescent probes is more feasible within the near-infrared (NIR) transparency window (NIRW: 650-900 nm) where hemoglobin and melanin absorbance significantly decreases and water absorbance is still low. Chromophores in genetically-encoded probes can be formed either autocatalytically from amino acids, as in a case of green fluorescent protein (GFP)-like proteins, or be bound to apoproteins. The most red-shifted fluorescent proteins (FPs) of the GFP-like family have excitation and emission spectra completely or partially outside of the NIRW and suffer from low brightness and modest photostability. Natural bacterial phytochrome photoreceptors (BphPs) utilize low molecular weight biliverdin as a chromophore. BphPs binding biliverdin provide many advantages over other chromophore containing proteins. First, unlike the chromophores of non-bacterial phytochromes, biliverdin is ubiquitous in mammals. This makes BphP applications in mammalian cells, tissues, and whole mammals as easy as conventional GFP-like FPs, without supplying chromophore through an external solution. Second, BphPs exhibit NIR absorbance and fluorescence, which are red-shifted relative to that of any other phytochromes, and lie within the NIRW. This makes BphPs spectrally complementary to other existing biophotonic tools such as all GFP- like FPs and available optogenetic tools. Third, independent domain architecture and pronounced conformational changes upon biliverdin photoisomerization make BphPs attractive templates to design various photoactivatable NIRFPs. Based on our analysis of the photochemistry and structural changes of BphPs we plan to develop three new types of the BphP-based NIRFPs. These include three bright and spectrally resolvable NIRFPs, putatively called short-, medium-, and long-NIRFPs (Aim 1); photoactivatable with non- phototoxic NIR light PA-NIRFPs that are initially dark but become fluorescent either in short-, medium-, or long- NIR spectral regions, and photoswitchable either irreversibly (PS-NIRFPs) or repeatedly (RS-NIRFPs) between these NIR regions (Aim 2); and NIR reporters for protein interactions and phosphorylation based on a reversible bimolecular fluorescence complementation approach utilizing monomerized versions of NIRFPs (Aim 3). We will apply directed molecular evolution approaches based on rational structure-based design and random mutagenesis of candidate proteins, followed by flow cytometry bacterial cell sorting, screening colonies on Petri dishes, and multiwell plate protein characterization. These conventional techniques will allow screening for standard FP properties such as excitation and emission wavelengths, overall brightness, photostability, pH-stability, and folding at physiological temperatures. New high-throughput screening methods will be developed to specifically optimize BphP-based NIRFPs. Selection of NIRFPs with high quantum yield, a crucial parameter for BphP-derived FPs, will be performed using time-resolved fluorescence lifetime measurements of thousands of colonies simultaneously. To screen for a high affinity to biliverdin, which does not penetrate through the inner bacterial membrane, a pulse-chase production of biliverdin using heme oxygenase co-expression and targeting of BphP NIRFPs to bacterial periplasmic space accessible for exogenous biliverdin will be employed. Promising NIRFP candidates will be directly screened in mammalian cells using shuttle vectors to optimize protein folding and stability in mammalian cells, affinity to endogenous biliverdin and low cytotoxicity. Optimized NIR probes will be tested in mouse tumor models and applied to studies in living mammals. The resulting NIR probes will extend fluorescence imaging methods to deep-tissue in vivo macroscopy including multicolor cell and tissue labeling, cell photoactivation and tracking, detection of enzymatic activities and protein interactions in mammalian tissues and whole animals.