NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence

Abstract

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

Figure 1

Timeline illustrating several significant events that occurred in the field of bioluminescence.

Figure 2

Bioluminescence is based upon a chemical reaction that occurs between the luciferase enzyme and corresponding substrate. (A) Firefly luciferase (FLuc) reacts with D-luciferin in the presence of adenosine triphosphate (ATP), molecular oxygen, and magnesium to produce light. (B) Both Renilla (RLuc) and Gaussia luciferase (GLuc) only require coelenterazine and oxygen to produce light. (C) Bioluminescence from the NanoLuc (NLuc) system occurs when the optimized substrate called furimazine reacts with NLuc in the presence of molecular oxygen. This reaction yields furimamide and luminescence output.

Figure 3

The NanoLuc luciferase (NLuc) technology has been successfully utilized for several applications, including the investigation of protein – protein and protein – ligand interactions, exploring gene regulation and cell signaling, monitoring protein stability, utilization as BRET-based biosensors, and bioluminescence imaging.

Figure 4

Quantitative assessment of protein interactions under physiological conditions using NanoBiT. NanoBiT was used to study the rapamycin-inducible FKBP rapamycin binding protein (FRB)/FK506 binding protein (FKBP) interaction mammalian cells, using FRB-11S and FKBP-114 fusion proteins. Adapted with permission from ref. . Copyright 2015 American Chemical Society.

Figure 5

Monitoring of gene expression with cell lines expression NanoLuc (NLuc) integrations. (A) HAP1 cells were stimulated with three cytokines, IFN-β, activin A, and FGF1 for 4, 8, or 24 h with a final concentration of 50 ng/mL. RNA was isolated and analyzed for IFIT1, DACT1, and EGR1 signatures. (B) Clonal cell lines bearing the NLuc integrations in IFIT1, DACT1, and EGR1 were stimulated with three cytokines. NLuc levels were measured at 24 h for IFIT1 and DACT1and after the noted time points for EGR1 from collected cell lines. Reproduced with permission from ref. . Copyright 2015 Nature Publishing Group.

Figure 6

NanoLuc (NLuc) fragments for determining protein aggregation. (A) Schematic of the aggregation assay in which a fusion of a protein to the N-terminus of NLuc was used to monitor protein aggregation, with decreased luminescent signal signifying protein insolubility. (B) Luminescence output from individual NLuc fragments and combined fragments 50 min after addition of furimazine. (C) As a proof-of-concept, individual or both NLuc fragments were expressed in mammalian cells. Luminescence required the presence of both NLuc fragments, N65, and 66C. Adapted with permission from ref. . Copyright 2016 American Chemical Society.

Figure 7

NanoBRET for detecting protein – protein interactions between Frb and FKBP. (A) In NanoBRET, there is a difference of 175 nm from NLuc to NCT. (B) BRET with RLuc8 and YFP was used as a control system and showed a 44 nm separation in signals. (C) Schematic of the NanoBRET system, in which two proteins were fused with NLuc or HaloTag and expressed in mammalian cells for in vitro BRET imaging. Adapted with permission from ref. . Copyright 2015 American Chemical Society.

Figure 8

Comparison of NLuc and GLuc for imaging of breast cancer. MDA-MB-231 cells were transfected with NLuc or GLuc. (A) Bioluminescence imaging of mice implanted in the mammary fat pad with MDA-MB-231 cells transfected with NLuc. After injecting furimazine, images were obtained with an open filter, 500 nm filter, and 520 nm filter. (B) Mice implanted with either NLuc or GLuc-expressing MDA-MB-231 tumor xenografts were injected with coelenterazine and imaged for two minutes. Adapted with permission from ref. . Copyright 2013 SAGE Publications.

Figure 9

Comparison of luminescence intensity of NLuc, FLuc, and RLuc at 10 min. Spectral profile of NLuc, RLuc, FLuc, and click beetle red luciferase (CBR), showing a broad range of emission peaks. NLuc and FLuc were compared for sensitivity to (C) temperature and (D) pH. (E) Luminescence measured over time after treatment with cycloheximide to inhibit protein synthesis, showing that NLuc-PEST (NLucP) signal quickly decreased, followed by FLucP. (F) NLuc was used to investigate regulated changes in p53 stability, by expressing p53 –NLuc in HEK293 cells. The addition of etoposide caused an accumulation of p53, which could be measured with furimazine. Adapted with permission from ref. . Copyright 2012 American Chemical Society.