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Review
. 2019 Mar 26:7:56.
doi: 10.3389/fbioe.2019.00056. eCollection 2019.

NanoBRET: The Bright Future of Proximity-Based Assays

Natasha C Dale  1   2   3 , Elizabeth K M Johnstone  1   2   3 , Carl W White  1   2   3 , Kevin D G Pfleger  1   2   3   4
Affiliations
Review

NanoBRET: The Bright Future of Proximity-Based Assays

Natasha C Dale et al. Front Bioeng Biotechnol. .

Abstract

Bioluminescence resonance energy transfer (BRET) is a biophysical technique used to monitor proximity within live cells. BRET exploits the naturally occurring phenomenon of dipole-dipole energy transfer from a donor enzyme (luciferase) to an acceptor fluorophore following enzyme-mediated oxidation of a substrate. This results in production of a quantifiable signal that denotes proximity between proteins and/or molecules tagged with complementary luciferase and fluorophore partners. BRET assays have been used to observe an array of biological functions including ligand binding, intracellular signaling, receptor-receptor proximity, and receptor trafficking, however, BRET assays can theoretically be used to monitor the proximity of any protein or molecule for which appropriate fusion constructs and/or fluorophore conjugates can be produced. Over the years, new luciferases and approaches have been developed that have increased the potential applications for BRET assays. In particular, the development of the small, bright and stable Nanoluciferase (NanoLuc; Nluc) and its use in NanoBRET has vastly broadened the potential applications of BRET assays. These advances have exciting potential to produce new experimental methods to monitor protein-protein interactions (PPIs), protein-ligand interactions, and/or molecular proximity. In addition to NanoBRET, Nluc has also been exploited to produce NanoBiT technology, which further broadens the scope of BRET to monitor biological function when NanoBiT is combined with an acceptor. BRET has proved to be a powerful tool for monitoring proximity and interaction, and these recent advances further strengthen its utility for a range of applications.

Keywords: BRET; CRISPR; NanoBRET; NanoLuc; Nluc; fluorophore; ligand binding.

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Figures

Figure 1
Figure 1
The principle of bioluminescence resonance energy transfer (BRET) for monitoring biological proximity. (A) The donor luciferase and acceptor fluorophore are not in close proximity (>10 nm), such that no resonance energy transfer occurs and there is no emission from the fluorophore. (B) The donor luciferase and acceptor fluorophore are in close proximity (<10 nm), allowing BRET to occur that reduces the donor light emission and results in light emission from the acceptor. When these BRET tags are fused to proteins or small molecules of interest, the non-radiative energy transfer from the donor luciferase to the acceptor fluorophore produces a change in the BRET ratio that in turn indicates proximity of the tagged proteins and/or small molecules.
Figure 2
Figure 2
Suitability of Nluc for BRET binding studies. (A,B) BRET ligand binding assays for transiently-transfected Rluc8-β2-adrenoceptor (β2AR) (A) and Nluc-β2AR (B) treated with increasing concentrations of alprenolol-TAMRA in the absence or presence of 10 μM unlabeled alprenolol. Data are mean ± s.e.m. of three experiments performed in quadruplicate. (C,D) Inhibition of the BRET signal for HEK293 cells stably-expressing Nluc-β2AR treated with 10 nM propranolol-BY630 (C) or propranolol-BYFL (D) and increasing concentrations of unlabeled ligands as shown. Each data point represents mean ± S.E.M. of five [all curves in (C) and propranolol in (D)] or four (D) separate experiments. In each experiment triplicate determinations for each data point were made. Raw BRET Ratio = (long wavelength emission/short wavelength emission), data presented on log10 scale. Reproduced from Stoddart et al. (2015).
Figure 3
Figure 3
Investigating recruitment of genome-edited β-arrestin2 using BRET. (A) Schematic representation of the exogenously expressed GPCR fused to Venus (exGPCR/Venus) and β-arr2/Nluc BRET configuration. HEK293FT cells expressing genome-edited β-arrestin2 fused to Nluc (geβ-arr2/Nluc) transiently transfected with cDNA coding for (B,C) CXCR4 fused to Venus (exCXCR4/Venus; red circles) or (D,E) V2R fused to Venus (exV2R/Venus, blue circles) as well as HEK293FT cells transiently co-transfected to express exogenous β-arrestin2 fused to Nluc (exβ-arr2/Nluc, black squares) at near endogenous levels and (B,C) exCXCR4/Venus or (D,E) exV2R/Venus. (B,D) Kinetic profiles of β-arrestin2/Nluc recruitment initiated by addition of CXCL12 (30 nM) or AVP (100 nM) for CXCR4 and V2R, respectively. Concentration-dependent recruitment of genome-edited or exogenous β arrestin2/Nluc to (C) exCXCR4/Venus or (E) exV2R/Venus mediated by CXCL12 (10 pM−100 nM) or AVP (10 pM−100 nM), respectively. Inserts (d,e) show expanded view of geβ-arr2/Nluc recruitment to exV2R/Venus presented in (D,E). Points and bars represent mean ± S.E.M. of three or four independent experiments. p[EC50] = –log10 half maximal effective concentration. Reproduced and modified from White et al. (2017) under a Creative Commons Attribution 4.0 International License. Full terms provided at http://creativecommons.org/licenses/by/4.0/.
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