Development of Genetically Encoded Bioluminescent Tools for Sensing and Manipulating Biological Processes

Bioluminescence has been a valuable method for visualizing biological activities in vivo. This technique is powered by the biochemical reaction of substrate (luciferin) oxidation by an enzyme (luciferase), producing detectable light. Bioluminescent photons can penetrate tissue and living organisms, providing sensitive and non-invasive means to monitor physiological activities. Historically, this approach has been employed primarily to track cells and gene expression trends, but recent innovations in probe design are broadening the range of observable phenomena. Notably, recent developments have integrated bioluminescence with optogenetic tools, allowing luciferase-luciferin reactions to activate light-sensitive proteins that lead to signal transduction and subsequent cellular functions. Despite the current advancements in bioluminescent tool development, there are still needs for better tools that will push the boundary of bioluminescence technology towards broader biological applications.
The overall objective of this dissertation is to create innovative bioluminescent tools for visualizing or manipulating biological processes, aiming to deepen our comprehension of these processes, with a focus on neurobiology. In the first project, I developed the first genetically encoded bioluminescent indicator for potassium ion (K+), a critical electrolyte for numerous physiological processes. I designed the first analyte sensory luciferin, potassiorin, that installs with a K+ binding moiety. I further engineered a luciferase, termed BRIPO, to work in conjugation with potassiorin. This novel system displayed excellent specificity and sensitivity towards K+, making it a robust tool for monitoring K+ dynamics in biological systems in real time, which was evidenced by comprehensive testing in various biological models, including cell lines, primary neurons. It also sets a foundation for developing other bioluminescent indicators by incorporating various sensory moieties into the luciferin structure, which could significantly enhance the study of cellular and molecular processes.
In my second project, I engineered orthogonal luciferase-luciferin pairs with distinct substrate availability and resolved emission profile. These pairs served as the foundation for creating orthogonal luminopsins (fusion proteins of luciferase and channelrhodopsin) capable of controling channelrhodopsin activation in response only to the corresponding luciferin. This breakthrough enables the manipulation of membrane potential and subsequently neural activities, with the potential to facilitate complex studies on neural circuits and brain functions.
In summary, the presented research endeavors to craft innovative bioluminescent tools for visualizing and manipulating biological processes, with an emphasis on neurobiology. These innovative tools open up the possibility to deepen our understanding of the intricate biological systems.