Engineering bacterial phytochromes for near-infrared imaging in mammals

描述（由申请人提供）：对深层组织发育、代谢和致病过程的无创监测将推动现代生物学的发展。在近红外（NIR）透明窗口（NIRW: 650-900 nm）内，荧光探针对活体哺乳动物的成像更为可行，此时血红蛋白和黑色素的吸光度显著降低，而水的吸光度仍然很低。基因编码探针中的发色团可以由氨基酸自催化形成，如绿色荧光蛋白（GFP）样蛋白，也可以与载脂蛋白结合。类红移荧光蛋白家族的大多数红移荧光蛋白（FPs）的激发和发射光谱完全或部分位于NIRW之外，亮度低，光稳定性不高。天然细菌光敏色素光感受器（BphPs）利用低分子量胆绿素作为发色团。BphPs结合胆绿素比其他含有发色团的蛋白质有许多优点。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无处不在。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gfp样fp一样容易，而无需通过外部溶液提供发色团。其次，BphPs表现出近红外吸收和荧光，相对于任何其他光敏色素的红移，并且位于NIRW内。这使得BphPs在光谱上与其他现有的生物光子工具（如所有GFP样FPs和光遗传学工具）互补。第三，独立的结构域结构和胆绿素光异构后明显的构象变化使BphPs成为设计各种光活化nirfp的有吸引力的模板。基于我们对bphp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。这包括三个明亮且光谱可分辨的NIRFPs，假定称为短期，中期和长期NIRFPs (Aim 1)；具有非光毒性NIR光可光激活性的PA-NIRFPs，最初是暗的，但在短、中或长NIR光谱区域变成荧光，并且在这些NIR区域之间可不可逆（PS-NIRFPs）或重复（RS-NIRFPs）光切换（Aim 2）；和NIR报告蛋白相互作用和磷酸化基于可逆的双分子荧光互补方法，利用单分子版本的NIRFPs（目的3）。我们将应用基于合理结构设计和候选蛋白随机诱变的定向分子进化方法，然后是流式细胞术细菌细胞分选，培养皿上筛选菌落，以及多孔板蛋白表征。这些常规技术将允许筛选标准FP特性，如激发和发射波长、总体亮度、光稳定性、ph稳定性和生理温度下的折叠。将开发新的高通量筛选方法来专门优化基于bphp的nirfp。选择具有高量子产率的NIRFPs，这是bphp衍生FPs的关键参数，将同时使用数千个菌落的时间分辨荧光寿命测量进行。为了筛选对胆绿素的高亲和力（胆绿素不能穿透细菌内膜），利用血红素加氧酶共表达和BphP NIRFPs靶向外源胆绿素可进入的细菌周质空间，采用脉冲追逐法生产胆绿素。利用穿梭载体直接在哺乳动物细胞中筛选有前途的NIRFP候选蛋白，以优化蛋白质在哺乳动物细胞中的折叠和稳定性、对内源性胆绿素的亲和力和低细胞毒性。优化后的近红外探针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荧光成像方法扩展到体内深层组织宏观观察，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中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.