NanoBRET: The Bright Future of Proximity-Based Assays

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.

Intracellular Ionic Strength Sensing Using NanoLuc

“…Furimazine has minimal background bioluminescence compared to other luciferins [ 28 ], thus, exhibiting a higher signal-to-background ratio. All these properties of NanoLuc have led to its utility in a variety of applications including resonance energy transfer-based protein–protein interaction determination (BRET), gene regulation, and monitoring protein stability in diseased conditions [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. In fact, a number of biosensing strategies have been devised based on the BRET phenomenon [ 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ].…”

pHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of Tumor Acidosis

“…To the best of our knowledge, the pHLuc reporter is the first genetically encoded pH-sensitive luminescent reporter that would provide a simple and inexpensive means to study the pH e of tumors in vivo. An advantage of utilizing BRET for pHLuc is that the reporter ultimately combines the benefits of the bright fluorophore signal and the low background of a luciferase (Dale et al, 2019;Kobayashi et al, 2019). Moreover, this reporter provides the advantage of dual-color imaging with the use of a single furimazine substrate, and allows for the monitoring of pH changes in small rodents.…”

Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications

“…Moreover, when used as a fusion reporter, NanoLuc is theoretically less likely to cause function-altering steric hindrance. This allows it to be used in BRET assays where the possibility of changing the function of a fusion structure can be a concern [ 198 ]. Comprehensive reviews on NanoBRET methodology and applications have been published recently [ 197 , 198 ].…”

“…This allows it to be used in BRET assays where the possibility of changing the function of a fusion structure can be a concern [ 198 ]. Comprehensive reviews on NanoBRET methodology and applications have been published recently [ 197 , 198 ]. NanoBRET is successfully used for studying protein–protein interactions [ 184 , 199 ], or investigations on receptor activation [ 190 , 199 , 200 ], oligomerization [ 201 ], conformational states [ 186 ], proximities [ 189 , 202 ], and receptor–ligand interactions [ 200 , 201 , 203 ].…”