Kenkyu Journal of Pharmacology ISSN : 2455-9237
Illuminating Biomolecular Interactions Using Recent Advances of Nanoluc-Based Bioluminescence Resonance Energy Transfer Technology
  • Qi Qi ,

    Department of Pharmacology, Emory University, Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322; e-mail: qi.qi@emory.edu ;Tel: +1-404-727-1335

  • Yu Yan ,

    Department of Pharmacology and Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA

Received: 23-12-2015

Accepted: 28-12-2015

Published: 30-12-2015

Citation: Yu Yan, Qi Qi (2015) Illuminating Biomolecular Interactions Using Recent Advances of Nanoluc-Based Bioluminescence Resonance Energy Transfer Technology. KJ Pharmacol 1: 100103

Copyrights: © 2015 Qi Qi, et al.,


Biomolecular interaction is a universal component and of key importance to most biological processes, which plays a pivotal role in pharmacology. In recent years, the landscape of biomolecular interaction significantly expanded due to advances in a number of ingenious biochemical and biophysical technologies. Bioluminescence resonance energy transfer (BRET) is a naturally occurred physical phenomenon, and has been engineered to monitor direct molecular interactions, due to its stringent distance requirement (≤ 10 nm) to allow efficient energy transfer. However, the weak signal and broad spectrum of conventional BRET donor significantly hindered its application. Recently, a surge of the development of novel luciferase, NanoLuc®, as BRET donor brings a new wave of exploring various molecular interactions. Herein, we provide a review of the recent advances of NanoLuc-based BRET technologies in studying biomolecular interactions, particularly in pharmacological applications.


Keywords: Bioluminescence Resonance Energy Transfer; Nanoluc Luciferase; Protein-Protein Interaction; Drug-Target Interaction; Intramolecular BRET Biosensor.


Bimolecular interactions are a fundamental physical components spanning across various biological processes such as micro- or macromolecular aggregations, protein behavior and function, and enzymatic reactions [1]. Detailed understanding of the molecular interactions will allow us to characterize protein complexes and signaling pathways underlying cellular processes related to human disease, and to characterize drug-target interaction for drug development. Traditionally, immunochemistry related methods, such as GST pull-down and co-immunoprecipitation, are employed for studying the biomolecular interactions. Recently, a number of ingenious techniques have been utilized to study the complex network, kinetic and dynamic information of molecular interactions in vitro or in vivo.


Resonance energy transfer (RET) is a physical mechanism describing energy transfer between two chromophores. The stringent requirement of small-distance proximity makes RET technologies, such as fluorescence or bioluminescence RET (FRET/BRET), become indispensable tools to study direct molecular interactions [2]. In the setup of BRET technology, the major unique feature is the energy donor which using intrinsic light from luciferase-luciferin enzymatic reaction as excitation, instead of the external laser excitation used in FRET. Briefly, BRET requires molecule of interest to be attached with donor and acceptor molecule, typically luciferase and fluorescent proteins, respectively. The donor and acceptor will come into close proximity upon molecular interaction. The energy transfer from enzymatic reaction induced luminescence to the corresponding acceptor will result in an increased emission at an acceptor-specific wavelength, generating the BRET signal. Compared to FRET, BRET attracted more attention due to its significant advantages in studying molecular interaction underlying biological processes in live organism, particular which is sensitive to the strong external laser excitation used in FRET. However, the weak signal and broad spectrum of conventional BRET donor significantly hindered its further application.


Nanoluc® luciferase (NLuc) is a novel luminescent protein innovated by Hall et al. [3] It is engineered from the luciferase of a luminous deep-sea shrimp, Oplophorus gracilirostris, and has been shown to be the smallest (19 kD) and brightest luciferase to date, with superior stability, glow-type luminescence and narrow emission spectrum. These superior properties make NLuc a promising candidate as a novel BRET donor. Indeed, extensive effort has been made to develop and apply NLuc-based BRET technology during the past year in 2015. Herein, we provide a review to summarize and compare different NLuc-based BRET configurations and their biological applications.


2. Selection of BRET acceptors paired with NLuc

In all NLuc-based BRET configurations, obviously, NLuc serves as BRET energy donor upon the oxidation of its substrate furimazine. In different configurations, various fluorophores have been recruited as BRET acceptors as shown in Table 1. With the emission peak of NLuc at 460 nm [3], the acceptors (Table 1) with excitation peak ranging from 484 to 595 nm, and emission peak ranging from 507 to 635 nm, have been validated as NLuc-compatible BRET acceptors. Generally, they could be summarized into two categories, small molecular dyes and auto-fluorescence proteins. Typically, small molecular dyes

need additional chemical labeling step in order to attach to the molecule of interest, such as HaloTag, SNAP tag or covalently conjugated with other small molecules. Additional washing step might needed to remove excess unbound dye molecules in order to eliminate the nonspecific energy transfer. Fluorescence proteins can be easily genetically fused to the N- or C-terminal of proteins of interest and serve as donor without additional labeling and washing step. The selection of acceptor has to be tested empirically to reach optimal signal-to-background ratio for different

Experimental purposes. Detailed experimental setup has been referred in individual literatures that will be discussed below.





Excitation peak

Emission peak

NCT dye{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}[6,13-15,18]{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}{C}

Small molecular dye

~595 nm

~635 nm


~560 nm

~575 nm


~550 nm

~570 nm


~555 nm

~580 nm


~630 nm

~650 nm


~503 nm

~512 nm


Fluorescent protein

~515 nm

~528 nm


~517 nm

~530 nm


~484 nm

~507 nm




































Table 1. Summary of NLuc-based BRET acceptors


3. Interrogation of protein-protein interaction in live cell using intermolecular BRET biosensors

The initial driving force in developing NLuc-based BRET technologies was to facilitate the interrogation of protein-protein interaction (PPI) study by leveraging the superior optical and biochemical properties of NLuc to enhance the sensitivity and push the limit of BRET technologies.

One of the major efforts has been made is using small molecular dye as BRET acceptor. The basic configuration relies on utilizing epitope tags, such as HaloTag [4-6] and SNAP-Tag [7], combined with their specific small molecular ligand as BRET acceptor. The configuration using HaloTag/NCT dye was developed by Promega and has been termed as NanoBRET [4]. Through Promega’s active collaboration with academic laboratory, NanoBRET has been utilized to study several biologically significant PPIs mainly involved in epigenetics [4-6], in conventional low-throughput fashion. Deplus et al. [6] employed NanoBRET technology to study the interaction between H3K4 methyltransferase SETD1A and Histone H3.3 as a functional readout of Ten-eleven translocation methylcytosine dioxygenase 2 and 3 (TET2/3) regulation of H3K4 methylation in live cellular context. With the same configuration, Clark et al. [5] and Machleidt et al. [4] established NanoBRET biosensors for interactions between bromodomain-containing proteins and histone proteins, and applied them to validate the cellular activity of known bromodomain inhibitors LP99 [5] and I-BET151 [4,8]. Kern et al. [7] used a slightly different strategy by employing SNAP-Tag combined with its small molecule ligand, BG-549 dye, as BRET acceptor. They developed a NLuc-based BRET biosensor to validate the hetero-dimerization between ghrelin receptor and dopamine receptor [7], and suggested a novel ghrelin receptor dependent dopamine receptor activation in the hippocampal structures.


Another major effort has been made in parallel is BRETn technology developed by Mo et al [9,10] as shown in Figure 1. In BRETn configuration, a yellow fluorescent protein Venus, which can be easily genetically engineered on the protein of interest, was used as BRET energy acceptor. BRETn system allows easy detection of PPI in an add-and-read fashion without additional labeling and washing steps that are necessary in HaloTag and SNAP-Tag based configuration. Mo et al. systematically compared and demonstrated the superior performance of BRETn over conventional BRET1 technology. They showed that BRETn is extremely sensitive in detecting PPIs of low abundancy at endogenous expression level. Such high sensitivity allowed miniaturization of BRETn technology in an ultra-high-throughput screening (uHTS) format, and thus largely pushed the limit of BRET technology in small molecule PPI inihibitor champion and large-scale PPI network mapping. As a proof of concept, Mo et al. discovered a first-in-class small molecular disruptor of proline-rich Akt substrate of 40 kDa (PRAS40) dimerization to reveal the physiological significance of PRAS40 dimer. Moreover, Mo et al. revealed and validated two novel PPIs through uHTS PPI profiling of the core proteins in Hippo signaling pathway, and suggested RASSF1 as a scaffold protein regulating Hippo signaling. As summarized in Figure 1, BRETn technology provides a robust platform allows sensitive PPIs detection in living cells for PPI mapping and PPI modulators discovery in a uHTS format.


4. NLuc-based BRET biosensor enables easy and sensitive detection of drug-protein interaction

Understanding the drug-protein interaction is the cornerstone of pharmacology. Current techniques requiring either purified system or radiolabeled material are practically undesirable for revealing drug-protein interaction details under a wide variety of conditions.



Figure 1. Schematic illustration of robust BRETn platform.



Stoddart et al. {C}[11]{C}{C} for the first time developed a series of intramolecular NanoBRET biosensors and demonstrated their potential application in monitoring drug-protein interactions, particularly using the ligand binding to G-protein-coupled receptors (GPCRs) as an example. These NanoBRET biosensors are consisted by fluorophore-labeled ligand and NLuc-tagged proteins. They used these NanoBRET biosensors to characterize the binding affinity of a panel of ligand to their specific GPCRs, such as -adrenergic receptor, adenosine receptor and angiotensin receptors, in both saturation and competitive binding assays in live cells without any additional washing and lysing steps. Similar specificity and parameters were obtained from these example studies compared with the data from previous conventional methods, suggesting NanoBRET a reliable technology for studying drug-protein interactions [12].


Robers et al. {C}[13]{C} utilized similar strategy and further extended this method to study other protein target classes, such as histone deacetylase and bromodomain-containing proteins. Noteworthy, similar as radio-labeled ligands, careful selection of labeling fluorophore is needed to minimize the labeling-dependent affinity shift.


5. Intramolecular NLuc-based BRET biosensors reveal protein dynamics

In contrast with the above mentioned BRET biosensors that enabling the study of intermolecular interactions, the intramolecular BRET biosensor configuration, by leveraging the protein conformational change between BRET on and off status upon protein in/activation, provides a unique avenue to study protein dynamics at various conditions.


Recently, Wang et al. {C}[14,15]{C}{C} used the principle of NanoBRET technology to decipher the mechanism of ERK1/2 phosphorylation of Rabin8, a guanine nucleotide exchange factor that regulates exocytosis. In order to test the hypothesis that Rabin8 adopts an autoinhibitory conformation upon ERK1/2 phosphorylation, Wang et al. constructed a Rabin8 intramolecular BRET sensor by tagging NLuc and HaloTag at its N- and C-terminal, respectively. In their hypothesized model, Rabin8 should undergo conformational change between closed inhibitory dephosphorylated conformation with low BRET signal and open active phosphorylated conformation with high BRET signal. Indeed, Wang et al. successfully observed that Rabin8 BRET sensor with kinase dead ERK2 had a significantly higher BRET signal than that with constitutively active ERK2, and consequently confirmed their hypothesized model that Rabin8 can inhibit its own catalytic activity by having the C-terminus block the guanine nucleotide exchange factor domain activity.


Besides monitoring protein dynamics, intramolecular BRET biosensor is also powerful to study drug-protein interactions. Johnsson et al. [16] for the first time invented luciferased-based indicators of drugs (LUCIDs). The key component underlying LUCIDs is an engineered ligand-Cy3-SNAP-NLuc-protein (LiCSNP) fusion construct. This LiCSNP construct can undergo conformational change from closed high BRET to open low BRET conformation upon competitive binding of external ligand with the intramolecular ligand to the receptor protein. By altering the proteins of interest (including dihydrofolate reductase, FKBP12, cyclophilin A, carbonic anhydrase II and DIG10.3), Johnsson et al. have demonstrated the validity of using engineered LUCIDs to monitor diverse drugs (including immunosuppressants, antiepileptics, anticancer and antirrhythmics agents) in patient samples with portable devices. By introducing a secondary tethered ligand in LUCIDs setup, Johnsson et al. [17] further developed a chemical ligand-associated steric hindrance (CLASH) approach to modulate protein activity using unrelated effectors.

Conclusion and Perspective

In 2015, NLuc-based BRET technologies have transformed the study of molecular interactions from diverse aspects, including protein-protein interaction, drug-protein interaction and engineered protein biosensors. This success is largely due to the advantages of the superior optical and biochemical properties of NLuc which enables the robust detection of molecular interactions in a physiological relevant context. The increased demand and expanded landscape of molecular interaction networks would encourage exploitation of the current and future potential of NLuc-based BRET technologies.


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