Orthogonal split luciferases for imaging multiplexed cellular behaviors

项目摘要 疾病状态的特征在于多种细胞类型之间发生的复杂相互作用， 生物体内的生物分子。研究人员已经转向生物发光成像（BLI）来跟踪 这些细胞的行为，并了解疾病的基础和有效的发展， 治疗。BLI利用生物发光酶的发光能力， 单个细胞而无需手术。因为生物体不发光，BLI非常敏感， 能够探测到老鼠体内的一个发光细胞。这使得研究人员 研究抗癌药物的有效性，感染的位置和进展，以及 干细胞治疗尽管BLI在该领域无处不在，但它同时具有多种不同细胞类型， 仍然困难。我们的研究旨在通过一种独特的方法， 多组分生物发光成像为了实现这一目标，我们将重新利用一个分裂的生物发光 一种叫做NanoBiT的蛋白质。NanoBiT包含由小肽组成的异二聚体结合对，称为 SmBiT和一个更大的蛋白质亚基，称为LgBiT。NanoBiT仅在SmBiT 和LgBiT结合。在目标1中，我们将使用蛋白质工程和定向进化技术 以产生正交SmBiT-LgBiT结合对。将克隆LgBiT酶的文库，并将一组 将合成SmBiT肽。高通量技术将评估每一个的光发射 LgBiT突变体与每种SmBiT肽的组合。“获胜”的突变体将通过下一个测序 第二代测序（NGS）和突变将被组合以形成优化的正交探针。在Aim中 我们将在哺乳动物细胞、组织模型和动物体内测试我们新的正交NanoBiT探针。 活老鼠首先，为了提高NanoBit光发射的组织穿透性，我们将调制 通过将小分子荧光团附加到我们的SmBiT肽来实现生物发光。稳定哺乳动物 含有我们的探针的细胞系接下来将在组织模型中用我们的SmBiT-荧光团探针进行测试， 在活的老鼠身上。探针将根据其灵敏度和选择性进行判断。这项工作将是第一次努力 使NanoBiT适用于多组分成像。我们的蛋白质工程数据将立即用于 生物发光成像社区。此外，这些探针将用于蛋白质-蛋白质成像， 宿主-病原体和癌症-免疫细胞相互作用。

PROJECT SUMMARY Disease states are characterized by complex interactions that occur between a variety of cell types and biomolecules in an organism. Researchers have turned to bioluminescence imaging (BLI) to track the behavior of these cells and understand the underpinnings of disease and the development of effective treatments. BLI utilizes the light-emitting ability of bioluminescent enzymes to sensitively illuminate individual cells without the need for surgery. Because organisms do not glow, BLI is exquisitely sensitive, with the ability to detect as few as one glowing cell in the body of a mouse. This has enabled researchers to study the efficacy of cancer-killing drugs, the location and progression of infection, and the success of stem cell treatments. Despite its ubiquity in the field, BLI of multiple different cell types simultaneously remains difficult. Our research seeks to further expand the utility of this tool through a unique approach to multicomponent bioluminescence imaging. To accomplish this, we will repurpose a split bioluminescent protein called NanoBiT. NanoBiT comprises a heterodimer binding pair made up of a small peptide, called SmBiT, and a larger protein subunit, called LgBiT. NanoBiT is only capable of light emission when SmBiT and LgBiT bind. In Aim 1 of the proposal, we will use protein engineering and directed evolution techniques to produce orthogonal SmBiT-LgBiT binding pairs. Libraries of LgBiT enzymes will be cloned, and a panel of SmBiT peptides will be synthesized. High-throughput techniques will evaluate the light emission of each LgBiT mutant in combination with each SmBiT peptide. "Winning" mutants will be sequenced via next generation sequencing (NGS) and mutations will be combined to form optimized orthogonal probes. In Aim 2 we will test our new orthogonal NanoBiT probes in mammalian cells, tissue models, and in the bodies of live mice. First, to improve the tissue penetration of NanoBit light emission, we will modulate the color of bioluminescence by appending small molecule fluorophores to our SmBiT peptides. Stable mammalian cell lines containing our probes will next be tested with our SmBiT-fluorophore probes in tissue models and in live mice. Probes will be judged by their sensitivity and selectivity. This work will represent the first effort to adapt NanoBiT for multicomponent imaging. Our protein engineering data will be immediately useful to the bioluminescence imaging community. Further, these probes will be useful for imaging protein-protein, host-pathogen, and cancer-immune cell interactions.