BRET, like FRET, is also used to investigate conformational changes and/or protein–protein interactions. However, BRET uses bioluminescent luciferase enzymes as donor molecules rather than fluorophores and does not require excitation from an external light source – a process that can cause photobleaching, autofluorescence, and light scattering. This means BRET has a very low background and increased signal-to-noise ratio compared to FRET (Dale et al., 2019). Renilla luciferase (RLuc), isolated from the Renilla reniformis sea pansy, was one of the first luciferase enzymes to be used in BRET (Dale et al., 2019). In the case of RLuc, the luciferase enzyme catalyses the ATP-independent oxidation of a luciferase substrate, coelenterazine (Dale et al., 2019). The luciferase donor molecule non-radiatively transfers energy to an acceptor fluorophore if the pair is in close proximity, in a manner similar to FRET (Figure 1B). One of the latest developments in BRET has been the introduction of a superior luciferase enzyme called Nanoluciferase (NLuc) (Dale et al., 2019). NLuc is a 19 kDa subunit derived from a larger, multi-subunit luciferase isolated from the deep-sea shrimp Oplophorus gracilirostris (Hall et al., 2012). NLuc has several advantages over RLuc: it is more stable; it is approximately 150 times brighter; and its smaller size means steric hindrance is less likely (Dale et al., 2019).

NLuc has been further exploited to produce an NLuc-derived split luciferase reporter system termed NanoBiT. NanoBiT is composed of two NLuc derived fragments: an 11 amino acid-long small bit (SmBiT) or high bit (HiBiT) fragment; and a second large bit fragment (LgBiT) (17.6 kDa). These fragments are genetically fused to separate parts of the same protein or two distinct proteins. When in proximity, the two fragments can reconstitute and produce a luminescent signal. HiBiT is commonly used for ligand binding assays (Soave et al., 2016, Stoddart et al., 2015), whereas SmBiT is better suited to investigate protein–protein interactions due to its lower affinity for LgBiT (Figure 1C) (Lohse et al., 2012).