UCLA

Protein nanocages are abundant throughout nature, comprising many different shapes, sizes, and functions. However, despite their wide variety, these assemblies share key features, including a modifiable exterior surface, interior volume, and subunit-subunit interfaces. These characteristics, in conjunction with general stability and monodispersity, confer on nanocages an incredible capacity for synthetic biology applications. Already, both natural and designed protein nanocages have demonstrated exciting potential in therapeutics, imaging, and materials. The primary focus of the dissertation is to explore the utility of protein nanocages as modular molecular delivery platforms.

In the first chapter, I briefly review prior works that have studied protein nanocages as potential molecular carriers and delivery vehicles. The most prominent natural cages featured in the literature, ranging from small ferritins to large virus-like particles, are discussed. These examples encompass applications such as small molecule loading and delivery to more complex chemical and/or genetic modifications. The emergence of designed nanocages as an alternative to evolved nanocages for synthetic biology is also discussed. However, despite these incredible advances, the general lack of specificity and modularity in protein cage-based encapsulation and delivery systems remains an ongoing challenge. In the second chapter, I present the ligand-operable cage (LOC), a new type of protein cage that can “open” in response to a specific ligand. These designs aim to address the lack of a modular nanocage platform with a target-based disassembly mechanism. Uniquely, the opening mechanism of LOCs relies on the spatial orientation of surface-fused adaptors. In this study, we fused designed ankyrin repeat proteins (DARPins) to the surface of two different nanocages, sulfur oxygenase-reductase and the previously designed T33-51 cage. We also developed two readout assays indicating successful nanocage disassembly: one based on fluorescence unquenching of an encapsulated fluorophore, and one based on a split-NanoLuc luciferase assay.

In the third chapter, I discuss the design of three new cages to: 1) demonstrate the modularity of the LOC platform, and 2) explore their ability to bind cancer targets. The LOC variants, all based on the same T33-51 core, were designed to bind either BARD1, BRCA1, or KRAS, which are all prominently implicated in cancer. We modeled the overall structure of these designs and their adaptor orientations using AlphaFold-based ColabFold. We discuss the synthesis and purification of these LOC variants, characterize their assembly, and show preliminary results from target-triggered disassembly experiments.

In the fourth chapter, I aim to develop a combinatorial DNA-triggered bioswitch incorporating a protein nanocage. In a prior study, a NanoLuc-Cy3 BRET-beacon was designed to turn “on” or “off” in response to an oligonucleotide (ODN) trigger. We sought to expand this existing modular bioswitch by conditionally encapsulating the target ODN (i.e., the bioswitch trigger) inside of a ligand-operable cage. In this manner, we could add an additional layer of modular control over the on-off state of the BRET-beacon. We detail the experimental scheme of this combinatorial switch, the synthesis of its components, and results from initial experiments.

In conclusion, the dissertation demonstrates the utility of protein nanocages as modular platforms for synthetic biology applications. Though prior works have exceptionally leveraged evolved cages, such as ferritin, VLPs, and other nonviral cages for molecular storage and delivery, we sought to address the general lack of a target-based, modular system. To this end, we demonstrate that our first LOC designs successfully released an encapsulated cargo upon specific target-binding using fluorescent and luminescent readout assays. Furthermore, we generated additional LOC variants that aimed to bind cancer-relevant proteins. Finally, we integrated a designed nanocage with a modular DNA sensor to create a combinatorial bioswitch. Taken together, these results, in consideration of experimental challenges and future directions, have exciting implications for future synthetic biology applications using protein nanocages.