Notes: This article describes the development of a new bridging immunoassay where NanoLuc® luciferase enzyme is used as an antibody label. (4901)

Notes: An insertional mutagenesis screen of the Dengue virus genome was used to identify regions tolerating small 15 amino acid insertions. The NS1 protein, which is essential for viral genome replication, was shown to be highly tolerant to insertions. NS1 was tagged with both a minimal fusion tag (Small BiT) and NanoLuc Luciferase. Tagged protein variants were assessed for viral infectivity, and used to investigate the protein localization and levels of NS1. (5075)

Notes: The development of NanoLuc Luciferase has led to a need for selective NanoLuc inhibitors to allow for bioluminescent suppression and multiplexing compatibility with existing assays. A lead compound with an IC50 of 600 nM against NanoLuc was further derivatized to create a family of potent and cell permeable inhibitors. These compounds were additionally developed to generate a second class of cell impermeable inhibitors. These inhibitors were tested for selectivity against Firefly luciferase and the NanoBiT system with no cross-reactivity.  (5106)

Notes: Activation of Notch isoforms via histone deacetylase inhibitors (HDACi) has been shown to suppress neuroendocrine (NE) cancers. Here, a novel HDACi called thailandepsin A (TDP-A) was characterized and shown to induce cell cycle arrest and apoptosis. The NanoBRET Target Engagement system was used to determine TDP-A binding to HDAC1. TT cells expressing an HDAC1-NanoLuc fusion were treated with TDP-A and competed HDAC tracer compound to determine in vivo binding affinity.  (5131)

Notes: Modified antisense oligonucleotides (ASOs) show increased delivery and stability in cells, however these modifications have off-target effects. Localization of ASOs to cytoplasmic ribonucleoprotein (RNP) granules is observed to be mediated by RNA binding proteins, FUS and PSF. These interactions are further investigated using the NanoBRET™ Protein-Protein Interaction System. NanoLuc^® tagged protein is produced using an in vitro transcription and translation system, purified, and bound to an acceptor oligonucleotide (AlexaFluor594-ASO). Various backbone and 2′ ASO modifications were screened for interaction with FUS truncations to determine the interaction domain. (5061)

Notes: The role of Polycomb-repressive complex 1 (PRC1) in spermatogenesis is investigated. RNF2, a component of PRC1, together with SALL4 is shown to bind the transcription start sites (TSSs) of genes involved in spermatogenesis. Further, a direct interaction between SALL4 and RNF2 is shown using coimmunoprecipitation. To validate a direct role of the SALL4-RFN2 complex in gene activation, regulatory elements were placed upstream of the NanoLuc luciferase and luminescence was monitored in a range of cell types. Together, a mechanism of gene activation is present where RNF2 activates transcription of sall4, and collectively they act on target genes. (5132)

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag^® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

Notes: Vascular endothelial growth factor (VEGF) receptor interactions are observed using the NanoBRET™ Protein-Protein Interaction System. A novel method of labeling VEGF at a single N-terminal cysteine (TMR) to maintain full activity is presented. NanoLuc^®-VEGFR2 and VEGF-TMR are used in conjunction to monitor an interaction and internalization into intracellular endosomes. The effect of receptor tyrosine kinase inhibitors such as Cediranib and vandetanib on internalization in living cells is assessed. (5060)

Notes: Adenomatous polyposis coli protein (APC) is shown to directly interact with HIV-1 Gag protein to stimulate Gag multimerization and spread of viral particles. Direct measurements of the Gag-Gag protein interaction were measured using the NanoBRET™ system. HeLa cells were co-transfected with Gag-HaloTag^® and Gag-NanoLuc^® expression vectors and BRET signal was monitored after 24 hours. ADC knockdown displayed a substantial decrease in viral production and Gag-Gag interaction. Together, the authors show ADC regulates Gag localization to the PM and viral packaging. (5058)

Notes: G protein coupled receptor (GPCR) oligomerization and protein interaction has been commonly investigated using bioluminescence resonance energy transfer (BRET). The need for exogenous expression of fusion proteins has been a short coming of the BRET assay. Here CRISPR/Cas9 mediated homology directed repair was used to generate protein-NanoLuc^® fusions under endogenous expression. The GPCR- β-arrestin2 interaction serves as a model of this system, where interaction and internalization are monitored under conditions where the donor luciferase endogenously expressed. (5059)

Notes: These authors used a NanoBiT® complementation assay to investigate interactions between proteins implicated in development of amyotrophic lateral sclerosis (ALS). Mutations in superoxide dismutase 1 (SOD1) and in TAR‐binding protein 43 kDa (TDP43) have been associated with ALS. The authors observed formation of SOD1 homodimers in neuronal cells transfected with SOD1 tagged with NanoBiT complementation partners, and found that SOD1 mutants associated with ALS were unable to form homodimers.  Next, possible interactions between SOD1 and TDP43 were assessed. When NanoBiT-tagged constructs of TBP43 and SOD were transfected into Neuro 2A cells, only NanoBiT-tagged SOD generated a luciferase signal. TBP43 did not self-associate to form homodimers, and SOD1 and TBP43 did not interact with each other to form heterodimers. The authors state that NanoBiT is a useful and sensitive tool for analyzing protein interactions under physiological conditions, and may provide a convenient way to monitor the modulation of SOD1 conformation by candidate treatments. (4765)

Notes: The authors characterize a novel orange-red fluorescent protein (CyOFP1), which serves as an efficient acceptor for resonance energy transfer from NanoLuc^® luciferase. An optimized fusion protein of CyOFP1 and NanoLuc^® (Antares) was demonstrated to function as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. During fluorometry, 50,000 cells in 10 μL Live Cell Imaging Solution were loaded into glass capillary tubes and gently pelleted by centrifugation for 15 s at 500g. 10 μL of Live Cell Imaging Solution with 20 μM D-luciferin, coelenterazine, ViviRen, or furimazine was then added. For BRET6 and Antares-expressing mice, tail vein injections were performed with 333 nmol (123 μg) of furimazine in 120 μL of 8% glycerol, 12% ethanol, 10% hydroxypropyl-β-cyclodextrin, and 35% polyethylene glycol 400 in water. (4760)

Notes: NanoLuc^® and Firefly luciferase reporters (pCMV128-NanoLuc and pCI-FireflyLuc) were co-transfected into HeLa cells to provide complementary measures of specific mRNA splicing and export activity. In the presence of splicing activity, firefly luciferase pre-mRNA is spliced in the nucleus, exported and translated resulting in firefly luciferase activity. In contrast, the open reading frame of the Nanoluc luciferase in pCMV128 NanoLuc^® was placed between two weak splice sites. If the pre-mRNA is spliced, the NanoLuc coding sequence is removed and no luciferase can be expressed. The NanoLuc^® reporter can only be translated when the unspliced pre-mRNA is transported to the cytoplasm. pCMV128 NanoLuc^® was generated by inserting the NanoLuc^® sequence into the NotI and BamHI sites of pCMV128, and then used for export assays. Cells were harvested 72 h post-transfection using 300 μl Passive Lysis Buffer. Lysates were analyzed using the NanoGlo® Dual-Luciferase^® Reporter Assay. (4744)

Notes: NanoLuc® Luciferase was used to monitor Plasmodium berghei throughout it's life cycle, including detecting single parasites in mosquitos, livers and asexual blood stages. (4922)

Notes: CRISPR/Cas9 editing was used to tag the GRP78 gene with NanoLuc^® luciferase in order to create a reporter of the intrinsic promoter activity of this gene. The authors used a single guide RNA against the N-terminal coding region of exon1 of the human GRP78 gene and constructed a donor gene that contains the puromycin-resistant gene following the NanoLuc^® gene. The NanoLuc^® gene was aligned with the puromycin-resistant gene through the 2A peptide sequence and was inserted into the pGL3-based vector (NL-2a-Puro-pGL3). To generate the donor gene, G78-NL, the N-terminal coding region that includes exon 1 of human GRP78 was inserted into the NanoLuc^® gene in the NL-2a-Puro-pGL3. After lysis of the cells expressing the indicated gene in 24- or 96-well plates with passive lysis buffer, the lysate was mixed with the NanoLuc^® substrate, and the luciferase activity in each well was measured using a GloMax^® luminometer. In some cases, the culture medium from each well was mixed with the NanoLuc^® substrate, and the activity for each was measured. (4754)

Notes: The authors used NanoLuc® Binary Technology (NanoBiT®) to build β-arrestin recruitment assays for two G-protein coupled receptors (CB1 and CB2) to create live-cell cannabinoid reporter assays. The assays were used to compare activity of synthetic cannabinoids and their metabolites and also to screen urine for CB receptor activity using the Nano-Glo® Live Cell Assay System. (4965)

Notes: Oligomerization of the glucose transporter (GLUT1) within this plasma membrane was assessed using the NanoBRET™ system. When expressed at high levels, GLUT1 has been shown to form tetrameric complexes with higher transport efficiency. Theoretical NanoBRET™ efficiency was determined for a variety of fluorescent protein acceptors and experiments were performed with mCherry as the NanoBRET™ acceptor. GLUT1 was shown to form a range of higher order complexes in live cells. Flow cytometry and immunoblotting were used in parallel to estimate GLUT1 density in cells. (5054)

Notes: Proteins fused to NanoLuc^® luciferase were used as the energy donor and AlexaFluor conjugated nucleic acids were used as the energy acceptor in BRET assays that measured protein:nucleic acid interactions. The assay was demonstrated with immunopurified proteins, cell homogenates, and in whole cells. NanoLuc^® fusion protein construction, expression, and purification were performed using the vectors pFN31K Nluc CMV-neo for amino-terminal clones and pFC32K Nluc CMV-neo for carboxy-terminal clones. BRET assays were performed in white 96-well plates. Alexa-linked anti-sense oligonucleotides at the indicated concentrations were incubated at room temperature for 15 min in 1X binding buffer with 106 RLU/well of immunoprecipitated NLuc fusion protein or whole cell lysate. Following the incubation, NanoGlo^® substrate was added at 0.1 μl/well. Readings were performed using a GloMax^® Discover System. (4757)

Notes: Nanoluciferase was used to characterize the conditions required to reliably perform single-cell BRET imaging of protein-protein interaction. HEK293T cells were cultured and transfected with vectors containing the Nluc gene sequence, such as is provided in the pNL1.1 Vector. Extracellular signal-related kinase (ERK) activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors, was detected by an Nluc-optimized BRET-based sensor of ERK activity. The authors report that the use of Nanoluciferase improved the resolution in terms of both time and space, enhanced the duration of signal stability, expanded the dynamic range and increased the sensitivity of the BRET signals in single-cell imaging. (4691)

Notes: The rAAV-mediated genome editing technology was used to introduce a NanoLuc^® reporter sequence downstream of and in frame with the last coding exon of the HIF1A gene in HCT116 cells. This created a reporter cell line with a single allele of HIF1A endogenously tagged with a NanoLuc^® reporter fusion. The cell line was used to study compound inhibition of Hif1a signaling. NanoLuc^® luciferase activity was measured using Nano-Glo^® reagent. For qHTS, cells at 1500 cells/well in 1536-well plates were incubated with test compounds at 37°C, 5% CO₂, 1% O₂ for 18 hours in a humidified CO₂ incubator with variable oxygen control, followed by addition of Nano-Glo^® reagent or CellTiter-Glo^® cell viability assay reagent. The HRE-bla assay was conducted using the CellTiter-Glo^® reagent. HCT116 and ME-180 cell proliferation was quantified as relative luminescence unit (RLU) values using the CellTiter-Glo^® viability assay reagent. (4761)

Notes: The receptor binding assays of the mature INSL5 and R3/I5 mutants were carried out using a NanoLuc-conjugated R3/I5 peptide as tracer and the human embryonic kidney (HEK) 293T cells transiently overexpressing human RXFP3 or human RXFP4 as receptor source. The receptor activation assays of the mature INSL5 and R3/I5 mutants were carried out using a cAMP-response element (CRE)-controlled NanoLuc^® reporter. Briefly, HEK293T cells were transiently cotransfected with the expression construct of human RXFP3 or human RXFP4 and the CRE-controlled NanoLuc^® reporter vector pNL1.2/CRE. (4756)

Notes: These authors designed a complementation reporter for detecting protein interactions under physiological conditions in mammalian cells. The engineered reporter (NanoBiT) is based on NanoLuc luciferase and is composed of two small subunits (18kDa and 1.3kDa), which associate weakly. The formation of a luminescent signal is dependent on the interaction characteristics of the proteins onto which the subunits are attached. The paper demonstrates utility of the NanoBiT assay for detecting protein interactions in live cells using  FRB/FKBP as an example, shows that response dynamics are rapid and reversible, and also illustrates use of the system to measure the pharmacology of kinase inhibitors that induce interaction between BRAF and CRAF. (4585)

Notes: This article details the advantages of NanoLuc® Luciferase in the scientific community. (4924)

Notes: The authors site-specifically conjugated the NanoLuc^® luciferase reporter to WT and mutant FGF2 proteins to create non-radioactive tracers that could be used to quantify ligand-receptor interactions with fibroblast growth factor receptors. Prior to Nanoluc^® conjugation, the biological activities of recombinant WT and mutant FGF2s were measured using receptor activation potency assays using a serum response element (SRE)-controlled NanoLuc^® reporter (pNL1.2/SRE) in HEK 293T cells expressing endogenous FGF receptor. Receptor-binding assays using the FGF2-Nanoluc^® tracers were carried out on overexpressed and endogenous FGF receptor in HEK293T cells. (4758)

Notes: A NanoLuc^® luciferase reporter was used to further understand the mechanism of Nrf2 inhibition of proinflammatory cytokine gene induction. Approximately 1 kb upstream region of the IL6 gene TSS containing the WT ARE (GCTGAGTCA) or mutated ARE (AGATCTGAC) was conjugated to the translation start site of the NanoLuc^® gene in the pNL2.2 vector. 293T cells (2.0 × 10³ cells per well) were plated in 96-well plates 24 h before transfection. The NanoLuc^® reporter vectors were co-transfected with pQC-FLAG-hNRF2T80R (constitutively active NRF2) plasmid33 and pcDNA3-p65 plasmid. After 48 hours of the transfection, the luminescence was quantified and normalized using NanoGlo^® Dual-Luciferase^® Reporter Assay. (4752)