Bioluminescence imaging is a powerful modality for probing cellular and molecular features in vivo. This technology involves a luciferase enzyme that oxidizes a small molecule luciferin, thereby producing light. The recent development of NanoLuc, a small, engineered luciferase with enhanced brightness and stability, has accelerated the adoption of bioluminescence imaging as a research tool. Significant strides have been made in both enzyme and substrate engineering to improve NanoLuc sensitivity as an in vivo probe. Furthermore, the NanoLuc toolbox has been expanded to include split reporters, circularly permutated variants, and probes for bioluminescence resonance energy transfer. These tools serve as the foundation for more intricate sensing architectures that cover a broad range of applications. Although advances in luciferase design have broadened the scope of bioluminescence, there remain key limitations. For example, bioluminescence has primarily been limited to tracking cell proliferation or gene expression in vivo. Few luciferases exist that can report directly on individual biomolecules or small molecule metabolites. Moreover, many of the existing platforms are not modular and difficult to modify for alternative analytes. Minor changes can result in large perturbations of the structure decreasing overall sensitivity and dynamic range. Thus, novel probes and sensors require substantial optimization through time-intensive empirical screening. Importantly, there are few generalizable bioluminescent platforms to visualize complex cellular events such as cell-cell contacts or RNA dynamics. The lack of modular and generally applied bioluminescent tools has slowed efforts to dissect biological networks. To address these limitations, I engineered and applied several new classes of bioluminescent tools. In one area, I developed a simple and modular NanoLuc sensor for proteases. I demonstrated that a glycine-serine peptide can be appended to the C-terminus of NanoLuc to decrease photon output. Insertion of a proteolytic cleavage sequence between the glycine-serine “cage” and NanoLuc allowed for sensing of proteases through protease dependent uncaging. I showcased the robustness of this sensor for detection of PPEP-1, a Clostridiodes difficile specific protease. A simple phone camera was used to capture signal from stool samples containing PPEP-1. This modular sensor can potentially be adapted for detection of other proteases by exchanging the proteolytic cleavage sequence through standard molecular biology techniques. In a second area, I developed two platforms that leveraged a split variant of NanoLuc to image dynamic cellular processes. The first system comprises a method to label and track interacting cells. One fragment of split NanoLuc is secreted by “sender” cells while the complementary piece is secreted by “receiver” cells. A single chain variable fragment (scFv)-peptide binding interaction was used to facilitate reassembly of the split domains. One split fragment was bound to the receiver cell, enabling localized light production on the cell surface. I demonstrated labeling of interacting cells in a time- and distance-dependent fashion. The second platform used split NanoLuc to detect and visualize RNA transcripts. The split domains were fused to two independent RNA binding proteins (RBP) that each recognize their own cognate RNA hairpin bait. Transcripts of interest were tagged with a novel RNA bait scaffold to facilitate localization of the RBP-split NanoLuc fusions. Binding of the RBP to the RNA bait enabled complementation of NanoLuc and light production for sensitive transcript detection. These new tools broaden the scope of bioluminescence imaging and set the stage for interrogating more diverse biological processes. My third project area involved the development of a luminescence-based microfluidic system to streamline future probe engineering. I optimized an existing optical train for fluorescence-based sorting to enable detection of dimmer luminescent probes. Using the redesigned optical train, luciferases could be detected at µM concentrations in nL to pL volumes. The microfluidic platform coupled with future chip designs will enable high-throughput screening of luciferase mutants in directed evolution campaigns. Such a technology will accelerate engineering efforts of brighter luciferases that can be further adapted for biosensing applications.

Background

Objective

Methods

Results

Conclusion

Trial registration number

Background

Methods

Results

Conclusions