Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate

Parts used

Key findings

• Directed evolution of the 19 kDa Oplophorus luciferase subunit through rational design and three phases of random mutagenesis generated NanoLuc with 16 amino acid substitutions. Starting with N166R stabilizing mutation, the first phase yielded C1A4E with 29,000-fold improvement in HEK293 cells. Final optimization with furimazine substrate achieved 2.5 million-fold luminescence enhancement over wild-type in mammalian cells with 150-fold higher specific activity than firefly or Renilla luciferases.
• Synthesis and screening of 24 novel coelenterazine analogues identified furimazine as optimal substrate for engineered luciferase variants. Furimazine produced 25-fold brighter luminescence than native coelenterazine with C1A4E variant and 30-fold improvement with NanoLuc. The substrate exhibited greater chemical stability in culture media and lower autoluminescence background than coelenterazine or coelenterazine h, with apparent Km of approximately 10 μM and glow-type signal half-life exceeding 2 hours at room temperature.
• NanoLuc functioned as transcriptional reporter with 80-fold higher signal than firefly luciferase, with PEST-appended variant showing 20-minute intracellular half-life enabling rapid response dynamics. Secreted NanoLuc retained activity in culture medium for 15+ hours at 37°C with 99% medium localization. Fusion to p53 detected DNA damage-induced stabilization with 15-fold response, while fusions to GR and PKCα enabled bioluminescence imaging of protein translocation with 1-5 second exposure times versus 3-10 minutes for beetle luciferases.

Abstract

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.