Dual Luciferase Assay for Secreted Luciferases Based onGaussiaand NanoLuc

Just recently, NanoLuc, a new engineered luciferase based on the small subunit of the luciferase from Oplophorus gracilirostris was introduced. Like the luciferase from Gaussia princeps, this luciferase is secreted into the medium. Both luciferases are the smallest and brightest luciferases known and well-suited for reporter assays. In our experiments, we demonstrate that both luciferases can be used together in a dual-reporter assay by solving the problem that NanoLuc produces a significant signal with coelenterazine, which is the substrate for Gaussia luciferase. We found that the background signal from NanoLuc with coelenterazine can be calculated from the determination of NanoLuc activity in the presence of its substrate furimazine. This in turn allows the precise determination of the activity of Gaussia which does not produce light in the presence of furimazine. Based on this observation, we developed a high sensitive dual secreted luciferase assay which allows the determination of both activities in a single cotransfection experiment. We demonstrate the versatility and robustness of the assay for the normalization of reporter gene activities. Since Gaussia luciferase and NanoLuc are nonhomologous reporters, the method to determine both luciferase activities may also be useful for coincidence reporter gene systems for high-throughput screening.