Reporter Assays and More: Applications of NanoLuc® Luciferase

NanoLuc® Luciferase brings exciting new possibilities and improvements to luminescence applications, including protein stability monitoring, detection of protein interactions using BRET, and in vivo imaging. This article highlights several recent papers that illustrate the use of NanoLuc® Technology as a sensitive reporter for challenging applications beyond those of classic bioluminescence reporter assays. Publication Date: June 2014; tpub 143 What is NanoLuc® Luciferase? In 2012, a new luciferase was developed that enhances and intensifies the properties of the natural enzyme from which it is derived. NanoLuc® Luciferase is smaller and much brighter than other luciferases, creating a much more sensitive analytical research tool. This enhancement brings new capabilities to bioluminescence-based research— extending and redefining the range of applications to which luciferase technology can be applied in the lab (1) . There are many distinct types of bioluminescence, all based on the interaction of the enzyme luciferase with a luminogenic substrate, generating light. Bioluminescent reporters provide advantages over fluorescent reporters because they provide a clear, quantitative signal over a wide concentration range and a low background signal. Reporter gene assays are the most well-known and widely used application of luciferase technology. Most reporter gene applications use firefly or Renilla (Sea Pansy) luciferase to study gene regulation and to serve as indicators of specific transactivation events. NanoLuc® Luciferase is a 19KDa enzyme engineered from a small luciferase subunit of the deep-sea shrimp Oplophorus gracilirostris. The enzyme and its substrate, furimazine, were designed together with the goal of generating improved luminescence in mammalian cells (1) . NanoLuc® Luciferase has a glow-type luminescence with a signal half-life greater than two hours and a specific activity 150-fold greater than that of either firefly or Renilla luciferases. Properties and Capabilities of NanoLuc® Luciferase The small size and extreme brightness of NanoLuc® Luciferase bring exceptional sensitivity to reporter assay applications—allowing detection at low expression levels (e.g., from endogenous promoters or in primary cells with poor transfection). These properties make NanoLuc® Luciferase an excellent alternative to firefly luciferase for reporter assays when working with low transfection efficiencies when firefly signals are too weak. The comparatively small size of NanoLuc® Luciferase may offer additional advantages when the molecular weight of firefly luciferase is problematic. In addition to these advantages for traditional reporter assays, NanoLuc® Luciferase brings increased sensitivity and improved performance characteristics to other luminescence applications, including measurement of protein stability dynamics, detection of protein interactions using BRET and in vivo imaging. © Promega Corporation, http://www.promega.com/pubhub 1 Brighter Signal, More Sensitive Reporter Figure 1. A comparison of the sensitivity of NanoLuc®, firefly and Renilla luciferase assays. The results in Figure 1 show that the signal from NanoLuc® Luciferase is two logs, or 100 times, brighter than the signal from either Firefly or Renilla luciferase. This translates to 100-fold greater sensitivity; therefore, 100-fold less protein is required to generate the same signal with NanoLuc® as with firefly or Renilla luciferases. This means improved detection at low expression levels, and much greater sensitivity—enabling detection of events at concentrations closer to physiological levels. NanoLuc® Luciferase fusions expressed at endogenous levels are bright enough to screen with high-throughput methods (2) , and genome editing techniques have been used to create NanoLuc® fusions to specific proteins of interest for studying certain disease pathways, allowing sensitive measurement of changes in endogenous levels of expression (3) . Engineered cell lines containing NanoLuc® fusions to specific proteins are commercially available from Horizon Discovery. The Advantages of a Small Reporter for Viral Packaging One research area where the small size of NanoLuc® Luciferase is proving an advantage is in the insertion of reporter genes into viruses where genome size is a limiting factor. Two recent reports describe use of NanoLuc® Luciferase to successfully create influenza and alphavirus reporters, respectively (4) (5) . Construction of influenza reporter viruses is complicated because the viral genome is small and all the viral genes are critical for infection. Therefore, replacement of an existing gene with a reporter gene or insertion of additional reporter sequences without affecting the ability of the virus to replicate and cause infection has proven difficult. To be successful, a reporter gene needs to be small enough to insert into the viral genome without eliminating any other vital functionality. Tran et al., (2013) describe use of NanoLuc® Luciferase to create a stable influenza reporter virus that retained virulence. Previous attempts to create influenza reporters with other luciferases resulted in attenuation of the virus and instability of the reporter gene. In © Promega Corporation, http://www.promega.com/pubhub 2 contrast, the NanoLuc® reporter virus displayed the same features as the wildtype virus in cell culture, was able to replicate in mice at a similar rate, and caused similar pathogenic effects. Bioluminescence imaging in live mice detected luminescence at two days post-infection, a detection level that was more sensitive than plaque assays. The bright signal allowed sensitive detection of the early stages of infection, and the small size allowed construction of a reporter in a situation where gene size was critical. In smaller viruses, it can be particularly difficult to introduce a reporter gene without compromising the ability of the virus to replicate and cause disease. Sun et al., (2014) compared the effectiveness of various NanoLuc® and firefly luciferase alphavirus reporter constructs. They assessed the ability of the luciferase genes to persist during infection of cultured cells in a mouse model and showed that the size and location of the reporter had a significant effect on successful replication and persistence. In vivo imaging of NanoLuc® and firefly constructs in mice showed that by 24 and 48h post-infection, the firefly luciferase signal was only detectable at the site of injection, but the NanoLuc® signal was detectable at many sites throughout the body and continued to increase up to 48 hours, indicating clearly the value of a stably integrated reporter with a bright signal. Improved Monitoring of Protein Stability NanoLuc® Luciferase is structurally stable and shows no cellular compartment bias in the absence of targeting sequences (1) . It can be used to monitor both the cellular location of fusion proteins and to monitor changes in intracellular protein abundance (6) (1) .