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Promega Corporation

Reporter Assays and More: Applications of NanoLuc® Luciferase

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Abstract

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.

Brighter Signal, More Sensitive Reporter

Figure 1. A comparison of the sensitivity of NanoLuc®, firefly and Renilla luciferase assays.

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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 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) .

Monitoring translocation of a NanoLuc® Fusion Protein using Bioluminescence Imaging (BLI). HEK293 cells expressing a Protein Kinase C (PKC)-NanoLuc® Luciferase fusion protein were measured for 20 minutes following PMA treatment and application of furimazine substrate. BLI was performed on an Olympus LV200 Bioluminescence Imager.

The intracellular level of many proteins is tightly controlled by a finely balanced rate of synthesis and degradation. Many proteins are maintained at low abundance by targeting for proteolysis via the ubiquitin/proteasome system. Certain conditions, such as cellular stress, result in increased protein stabilization, causing accumulation and subsequent activation of downstream signaling events. Control of protein stability is a primary mechanism for regulating many adaptive stress responses, and the ability to monitor changes in intracellular protein abundance is valuable for understanding the cellular response to specific chemical or environmental insults.
The short video below illustrates how NanoLuc® Luciferase can be used to monitor changes in intracellular protein dynamics, using a NanoLuc®-p53 fusion experiment from Hall et al., (2012) as an example.

Detecting Protein Interactions Using BRET

The study of protein interactions with BRET relies on the transfer of energy from luciferase (BRET donor) attached to one of the interacting partners, to a fluorescent BRET acceptor tagged to the other interacting protein or ligand partner. This energy transfer is moderated by the proximity of the two partners. NanoLuc® Luciferase offers unique advantages as a BRET donor, again because of its very bright light output and small size. The small size allows creation of fusion proteins where the chance of interference with the native protein function is minimized compared to larger fusion proteins. The brightness overcomes some key problems associated with BRET assays using Renilla luciferase.

Important characteristics for BRET applications are: Donor emission must overlap with acceptor excitation spectra; Donor and acceptor must be in close proximity (<10nm); Acceptor emission must be discernable from the donor emission, and; Output intensity is dependent upon the donor intensity. Donor brightness is therefore a key limiting factor in current BRET technologies. With a less bright donor such as Renilla luciferase, more spectral overlap between the donor and acceptor is required to get a good BRET signal, but this means higher background and less distinction between donor and acceptor signals. Because NanoLuc® Luciferase is so bright, it needs less spectral overlap with the acceptor in order to generate a signal, and more spectral separation between the donor and acceptor signals is possible.

The example below illustrates the benefit of NanoLuc® Luciferase as a BRET donor using the interaction between the proteins FKBP and FRB, moderated by rapamycin as a proof-of-concept (Figure 2). In this example, FKBP is labeled with a HaloTag® fusion protein attached to a fluorescent ligand optimized for NanoLuc®
-BRET applications (NanoBRET™ ligand). Its interacting partner, FRB, is labeled with NanoLuc® luciferase. In the presence of rapamycin, the proteins interact, causing excitation of the NanoBRET™ fluorescent ligand. Using Renilla luciferase as the donor, the problems of spectral overlap and low signal are apparent.

Figure 2. Comparison of the performance of NanoLuc® and Renilla luciferases in a BRET assay, Panel A. Schematic showing experimental principle. The FKBP12-rapamycin complex binds directly to the FKBP12-Rapamycin Binding (FRB) domain of mTOR in the presence of rapamycin. FKBP is labeled with a HaloTag® fusion protein attached to the fluorescent NanoBRET™ ligand. FRB was labeled with NanoLuc® or Renilla luciferase. Panel B. Interaction between FKBP and FRB as detected by BRET in the presence of increasing concentrations of rapamycin.

Panel A. Schematic showing experimental principle. The FKBP12-rapamycin complex binds directly to the FKBP12-Rapamycin Binding (FRB) domain of mTOR in the presence of rapamycin. FKBP is labeled with a HaloTag® fusion protein attached to the fluorescent NanoBRET™ ligand. FRB was labeled with NanoLuc® or Renilla luciferase. Panel B. Interaction between FKBP and FRB as detected by BRET in the presence of increasing concentrations of rapamycin.

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In a 2013 EMBO Journal paper, Deplus et al., (7) used NanoLuc® Luciferase in a BRET assay to help determine the role of the Ten-Eleven Translocation (TET) family of DNA-modifying enzymes in interacting with a histone methyltransferase complex associated with active chromatin. These authors identified an interaction between TET proteins and O-GlcNAc transferase (OGT), which catalyzes the addition of N-acetylglucosamine (GlcNAc) to numerous transcription factors, regulatory proteins and histone partners. Protein pull-down data showed that host cell factor 1 (HCF1), a known component of the H3K4 methyltransferase complex known as SET1/COMPASS and a known target for GlyNAcylation, also interacted with TET2/3 and OGT.

The authors used BRET to assess whether TET/OGT activity affected the interaction of SET1/COMPASS with chromatin by using NanoLuc® Luciferase fused to the H3K4 methyltransferase SETD1A as the energy donor and a fluorescently labeled histone H3.3-HaloTag® fusion protein as the energy acceptor. Close interaction between the SETD1A-NanoLuc® protein and H3.3 protein resulted in energy is transfer to H3.3, which was detected as light emitted by the fluorescent label. When cells expressing the fusion proteins were treated with an OGT inhibitor the BRET signal decreased. The BRET experiments helped confirm that TET2/3-OGT activity was necessary for SET1/COMPASS complex function and showed that TET and OGT activities promote binding of SETD1A, a component of the SET1/COMPASS complex, to chromatin.

In Vivo Imaging Applications of NanoLuc® Luciferase

Bioluminescent imaging of cells and molecular processes in whole animals is another area where the bright signal and low background of the NanoLuc® reporter and substrate provide more sensitive detection. In addition to the examples of imaging the spread of NanoLuc® labeled viral infections (4) (5) , NanoLuc® Luciferase has been used together with firefly luciferase to perform dual-luciferase molecular imaging studies of tumor growth in mice (8) . Stacer et al (2013) compared NanoLuc® Luciferase to another small, ATP-independent enzyme (Gaussia Luciferase) used for in vivo imaging and established that NanoLuc® Luciferase could be used successfully to image superficial tumors and internal organs in mice. The NanoLuc® signal increased in proportion to tumor growth, and secreted NanoLuc® could be detected in serum samples. They also combined NanoLuc® with firefly luciferase and demonstrated detection of two distinct steps in TGFb signaling in a dual-assay in both cell culture and whole animals.

Summary

NanoLuc® Luciferase is an ATP-independent, engineered luciferase that, together with its substrate furimazine, exhibits an extremely bright, glow-type luminescence with a signal half-life >2 hours. The combination of its small size and bright luminescence give NanoLuc® Luciferase unique properties that bring exceptional sensitivity to traditional reporter applications and enable inclusion of reporters in situations where size is critical—such as viral packaging. This article highlights several papers published over the last two years that demonstrate the utility of NanoLuc® Luciferase as a fusion partner for monitoring protein stability and translocation, as a BRET donor to provide better detection of protein interactions, and as a sensitive reporter for imaging events in cells and in living animals.

References

  1. Hall, M.P. et al. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol.
  2. Grooby, S. et al. (2012) XMAN™ NanoLuc™ Reporter Cell Lines: Application to Drug Discovery
  3. Morrill, P. et al. (2012) New Cellular Reporter Technologies using X-MAN™ Cell Models
  4. Tran, V., Moser, L.A., Poole, D.S., and Mehle, A. (2013) Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. J. Virol. 87 (24), 13321-9.
  5. Sun, C., Gardner, C.L., Watson, A.M., Ryman, K.D., and Klimstra, W.B. (2014) Stable, High-Level Expression of Reporter Proteins from Improved Alphavirus Expression Vectors To Track Replication and Dissemination during Encephalitic and Arthritogenic Disease. J. Virol. 88(4), 2035-2046.
  6. Robers, M., Binkowski, B., Hartnett, J., Wilkinson, J., Zimprich, C., Stecha, P. and Cong, M. (2014) Measuring Intracellular Protein Lifetime Dynamics Using NanoLuc® Luciferase. PubHub 2/2014; tpub 139,
  7. Deplus, R. et al. (2013) TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS. EMBO J. 32, 645–55.
  8. Stacer, A.C., Nyati, S., Moudgil, P., Iyengar, R., Luker, K.E., Rehemtulla, A., and Luker, G.D. (2013) NanoLuc reporter for dual luciferase imaging in living animals. Mol. Imaging 12(7), 1-13.

Figures

Figure 1. A comparison of the sensitivity of NanoLuc®, firefly and Renilla luciferase assays.

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Figure 2. Comparison of the performance of NanoLuc® and Renilla luciferases in a BRET assay, Panel A. Schematic showing experimental principle. The FKBP12-rapamycin complex binds directly to the FKBP12-Rapamycin Binding (FRB) domain of mTOR in the presence of rapamycin. FKBP is labeled with a HaloTag® fusion protein attached to the fluorescent NanoBRET™ ligand. FRB was labeled with NanoLuc® or Renilla luciferase. Panel B. Interaction between FKBP and FRB as detected by BRET in the presence of increasing concentrations of rapamycin.

Panel A. Schematic showing experimental principle. The FKBP12-rapamycin complex binds directly to the FKBP12-Rapamycin Binding (FRB) domain of mTOR in the presence of rapamycin. FKBP is labeled with a HaloTag® fusion protein attached to the fluorescent NanoBRET™ ligand. FRB was labeled with NanoLuc® or Renilla luciferase. Panel B. Interaction between FKBP and FRB as detected by BRET in the presence of increasing concentrations of rapamycin.

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