An Orthogonal Bioluminescent Platform For Multiplexed Imaging And Control

PROJECT SUMMARY In this project, we will engineer bright, small luciferases with bioavailable substrates orthogonal to NanoLuc/furimazine—the brightest and most widely employed bioluminescent system currently available to researchers. Bioluminescence is rapidly gaining traction as a general means to observe and control biochemical and electrophysiological signals in cells and is particularly advantageous for tracking and manipulating cells non- invasively in intact animal models of spinal cord injury, neurodevelopment, and neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic Lateral Sclerosis (ALS). The addition of one or more small, bright, independently controllable luciferin-luciferase systems will enable far more informative studies of the complex dynamics of the brain and other tissues. Here, we will take inspiration from the naturally orthogonal luciferin, vargulin (derived from marine ostracods), which produces very bright bioluminescence in its native context. We will take advantage of the small and bright coelenterazine-consuming luciferase variant SSLuc (derived from Oplophorus shrimp), an enzyme engineered by the project team. By making stepwise changes to the side groups of the luciferin coupled with directed evolution of the luciferase to increase specific activity, we will evolve SSLuc into an enzyme that efficiently generates light from vargulin rather than coelenterazine. In parallel, we will generate membrane-permeant caged analogs of vargulin that enable it to be delivered efficiently into cells in the central nervous system, along with caged analogs tailored for delivery to cells expressing specific enzymes, extending the utility of the vargulin platform. Our work is highly innovative, as it combines a unique blend of chemical synthesis and protein engineering to fill a long-standing void in bioluminescence-based imaging and cell manipulation. The proposed research is significant, as the bioluminescent tools will enable the direct and independent interrogation of multiple cells and pathways using bioluminescence, which is not currently possible with existing toolsets. The luciferin substrates generated in this work will be highly bioavailable, stable, and genetically targetable. Additionally, like other imaging technologies, the bioluminescent probes developed in this project will likely catalyze new discoveries in a broad spectrum of fields.