Orthogonal split luciferases for imaging multiplexed cellular behaviors

项目总结 疾病状态的特征是发生在各种类型的细胞和 生物体中的生物分子。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灵敏地照明 单独的细胞，而不需要手术。因为生物体不发光，所以BLI非常敏感， 能够探测到老鼠体内最少的一个发光细胞。这使得研究人员能够 目的：研究抗癌药物的疗效，感染的部位和进展，以及fi治疗的成功。 干细胞治疗。尽管它在fi领域无处不在，但同时具有多种不同细胞类型的BLI 仍然是fi邪教。我们的研究试图通过一种独特的方法来进一步扩大这一工具的实用性 多组分生物发光成像。为了实现这一点，我们将重新调整分离式生物发光体的用途 一种名为纳米比特的蛋白质。Nanobit由一对异源二聚体结合，该结合对由一种称为 SmBiT和一个更大的蛋白质亚基，称为LgBiT。纳米位只有当SmBiT时才能发光 和LgBiT绑定。在提案的目标1中，我们将使用蛋白质工程和定向进化技术 以制备正交的SmBiT-LgBiT结合对。LgBiT酶的文库将被克隆，一个小组 将合成一系列SmBiT多肽。高通量技术将评估每个 LgBiT突变体与每个SmBiT多肽结合。获胜的突变体将通过NEXT进行测序 世代测序(NGS)和突变将结合在一起，形成优化的正交探针。在AIM 2我们将在哺乳动物细胞、组织模型和人的体内测试我们的新的正交纳米比特探针。 活老鼠。首先，为了提高纳米比特光发射的组织穿透性，我们将调制 通过将小分子fl载体添加到我们的SmBiT多肽上进行生物发光。稳定的哺乳动物 含有我们的探针的细胞系接下来将在组织模型和组织模型中用我们的SmBiT-fluoropore探针进行测试 在活着的老鼠身上。探测器将根据它们的灵敏度和选择性进行判断。这项工作将代表fi的第一次努力 使纳米比特适用于多组分成像。我们的蛋白质工程数据将立即对 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，以及癌症-免疫细胞的相互作用。

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.