NanoBRET—A Novel BRET Platform for the Analysis of Protein–Protein Interactions

Abstract

Dynamic interactions between proteins comprise a key mechanism for temporal control of cellular function and thus hold promise for development of novel drug therapies. It remains technically challenging, however, to quantitatively characterize these interactions within the biologically relevant context of living cells. Although, bioluminescence resonance energy transfer (BRET) has often been used for this purpose, its general applicability has been hindered by limited sensitivity and dynamic range. We have addressed this by combining an extremely bright luciferase (Nanoluc) with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The small size (19 kDa), high emission intensity, and relatively narrow spectrum (460 nm peak intensity) make Nanoluc luciferase well suited as an energy donor. By selecting an efficient red-emitting fluorophore (635 nm peak intensity) for attachment onto the HaloTag, an overall spectral separation exceeding 175 nm was achieved. This combination of greater light intensity with improved spectral resolution results in substantially increased detection sensitivity and dynamic range over current BRET technologies. Enhanced performance is demonstrated using several established model systems, as well as the ability to image BRET in individual cells. The capabilities are further exhibited in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells.

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone

“…Alternatively, more redshifted fluorescent acceptors could be used, either red fluorescent proteins or synthetic fluorophores such as Cy3 or NCT. The latter have been successfully employed in combination with NanoLuc in various cell-based screening assays [22][23][24] and in the work by Johnsson. 25,26 Such synthetic fluorophores could be incorporated using bioorthogonal ligation reactions with non-natural amino acids or using self-labeling protein domains such as SNAP, CLIP or Halo-tags.…”

“…NanoLuc is a relatively small but stable protein that produces glow-type blue luminescence and has been reported to be 100-fold brighter than other luciferases. 18 NanoLuc thus represents an attractive donor luciferase for BRET-based assays, [22][23][24] as was recently successfully demonstrated by Johnsson and coworkers for monitoring low molecular weight therapeutic compounds. 25,26 To generate an efficient BRET sensor for antibody detection, we fused NanoLuc via a semi-flexible linker to mNeonGreen, one of the brightest monomeric fluorescent proteins currently available.…”

Engineering BRET-Sensor Proteins

“…However, the relatively long tail in the emission peak above 500 nm also allows it to be used as a BRET donor for orange/red fluorescent proteins and red-shifted dyes. NanoLuc, thus, represents an attractive donor luciferase for BRET-based assays, either in screening for protein-protein interactions or when used in BRET-sensors (Griss et al, 2014;Machleidt et al, 2015;Robers et al, 2015;Schena, Griss, & Johnsson, 2015;Stoddart et al, 2015). In a recent study, Perroy and coworkers directly compared the performance of RLuc8 and NanoLuc as BRET donors in a BRET-sensor for ERK activity.…”

“…Finally, fluorescence is ill-suited for use in optogenetics or cells that are endogenously light sensitive. In principle, all of these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET) (Bacart, Corbel, Jockers, Bach, & Couturier, 2008;De, Arora, & Jasani, 2014;Machleidt et al, 2015;Paley & Prescher, 2014). In these sensors, the donor fluorescent domain is replaced by a luciferase enzyme, which generates light by catalyzing the oxidation of specific organic substrate molecules.…”

“…Next, important design considerations will be discussed in more detail by describing the development of different classes of BRET-sensors, both from our own work and that of others. These examples are all based on the NanoLuc luciferase, a bright and very stable blue light-emitting luciferase developed by Promega that has quickly risen to prominence in the bioluminescence field (Dixon et al, 2016;England, Ehlerding, & Cai, 2016;Hall et al, 2012;Machleidt et al, 2015;Stoddart et al, 2015).…”