Fluorescent Protein Chromophore-Based Luciferins for Bioluminescence Imaging

Bioluminescence is routinely used to track cellular and molecular features in vivo. This technique relies upon the enzymatic oxidation of a small molecule to produce a photon of light. However, most bioluminescent probes exhibit suboptimal tissue penetrance, limiting applications in some preclinical models. We aimed to develop red-shifted tools for more sensitive, deep tissue imaging. Toward this end, we were inspired by the cell-compatible and red-emitting chromophore present in common fluorescent proteins (FPs). We synthesized two firefly luciferin analogues (FPLucs) based on the fluorescent motif. The probes produced >650 nm light, with peak emission values of 701 and 699 nm, making them amenable for tissue imaging. We further identified more optimal luciferases for processing FPLucs, using a combination of Rosetta-guided design and screening. When incubated with the analogues, the engineered luciferases exhibited improved light outputs compared to native firefly luciferase. The designer luciferase-luciferin pairs could also be readily detected in tissue mimics. Continued development of these and other fluorophore-inspired luciferins will expand applications of bioluminescence imaging.