Orthogonal split luciferases for imaging multiplexed cellular behaviors

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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.