In 2012, a new reporter enzyme called NanoLuc® Luciferase (NLuc) and its detection reagent, Nano-Glo® Luciferase Assay System, were introduced. NLuc was molecularly evolved from a 19kDa deep sea shrimp luciferase to increase stability and improve brightness. The natural substrate of the original luciferase, coelenterazine, was chemically evolved to a brighter, more stable substrate, furimazine. Together, NLuc and furimazine produce a luminescent signal >100-fold brighter than either firefly luciferase (FLuc) or Renilla luciferase (RLuc). The bonus is that the NanoLuc® luminescent signal has a long lifetime, exhibiting glow kinetics rather than a rapid flash signal like other small, bright luciferases such as Gaussia luciferase. Additionally, other key features of NLuc made it an ideal reporter. The small 19kDa enzyme was thermally stable, active over a broad pH range and required no post-translational modifications or maturation after translation to make an active enzyme. NLuc produces blue light with maximum emission at 460nm. To learn more, details of the creation, characterization and early applications of NLuc and Nano-Glo® Assay System are found in Hall et al. (1) .
Luciferase enzymes are an important tool for making recombinant reporter viruses, which are used to further understanding of viral life cycles and lethality in animal models (2) . Though luciferases come with their challenges, viruses with embedded reporters make it easier to follow infection in the same animal over time using bioluminescent imaging. FLuc is most commonly used for in vivo animal imaging due to its emission at ~600nm in live animals, which is better for deep-tissue imaging. At 61kDa, FLuc is a rather large protein, and limitations on viral capsid size mean that many viral genomes cannot incorporate an additional 1,600+ nucleotides of DNA. Viruses are more tolerant of the 34kDa RLuc reporter, but imaging is difficult because its blue light emission is easily scattered within animals. While imaging the blue light of NLuc does present a challenge in animal models, the brightness and long lifetime of the signal, as well as its small size, are significant benefits. Gaussia luciferase is also a small, bright enzyme, but is challenging to work with in vivo due to secretion from the cell and a rapid flash signal.
This paper will review two examples of how the NLuc size produced better, more stable recombinant viruses. See Table 1 for a more extensive list of NLuc-containing viruses or virus-like particles that have been cited in peer-reviewed publications. Recently, a short 11-amino acid bioluminescent protein tag was introduced that uses structural complementation to achieve a functional luciferase, which also can be applied to viral research. A review of two early papers using this peptide tag, known as HiBiT, follows the section concerning full-length NLuc.