Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Emerging Synthetic Bioluminescent Reactions for Non-Invasive Imaging of Freely Moving Animals.

Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin-luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.

A suicidal and extensively disordered luciferase with a bright luminescence.

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

A pH-sensitive closed-loop nanomachine to control hyperexcitability at the single neuron level.

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.

Low-cost and sustainable smartphone-based tissue-on-chip device for bioluminescence biosensing.

Several organ-on-chip and cell-on-chip devices have been reported, however, their main drawback is that they are not interoperable (i.e., they have been fabricated with customized equipment, thus cannot be applied in other facilities, unless having the same setup), and require cell-culture facilities and benchtop instrumentation. As a consequence, results obtained with such devices do not generally comply with the principles of findability, accessibility, interoperability, and reusability (FAIR). To overcome such limitation, leveraging cost-effective 3D printing we developed a bioluminescent tissue on-a-chip device that can be easily implemented in any laboratory. The device enables continuous monitoring of cell co-cultures expressing different bioluminescent reporter proteins and, thanks to the implementation of new highly bioluminescent luciferases having high pH and thermal stability, can be monitored via smartphone camera. Another relevant feature is the possibility to insert the chip into a commercial 24-well plate for use with standard benchtop instrumentation. The suitability of this device for 3D cell-based biosensing for monitoring activation of target molecular pathways, i.e., the inflammatory pathway via nuclear factor kappa-B (NF-κB) activation, and general cytotoxicity is here reported showing similar analytical performance when compared to conventional 3D cell-based assays performed in 24-well plates.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Maintain the light, long-term seasonal monitoring of luminous capabilities in the brittle star Amphiura filiformis.

The European brittle star Amphiura filiformis emits blue light, via a Renilla-like luciferase, which depends on the dietary acquisition of coelenterazine. Questions remain regarding luciferin availability across seasons and the persistence of luminous capabilities after a single boost of coelenterazine. To date, no study has explored the seasonal, long-term monitoring of these luminous capabilities or the tracking of luciferase expression in photogenic tissues. Through multidisciplinary analysis, we demonstrate that luminous capabilities evolve according to the exogenous acquisition of coelenterazine throughout adult life. Moreover, no coelenterazine storage forms are detected within the arms tissues. Luciferase expression persists throughout the seasons, and coelenterazine's presence in the brittle star diet is the only limiting factor for the bioluminescent reaction. No seasonal variation is observed, involving a continuous presence of prey containing coelenterazine. The ultrastructure description provides a morphological context to investigate the green autofluorescence signal attributed to coelenterazine during luciferin acquisition. Finally, histological analyses support the hypothesis of a pigmented sheath leading light to the tip of the spine. These insights improve our understanding of the bioluminescence phenomenon in this burrowing brittle star.

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

KPC-luciferase-expressing cells elicit an anti-tumor immune response in a mouse model of pancreatic cancer.

Mouse models for the study of pancreatic ductal adenocarcinoma (PDAC) are well-established and representative of many key features observed in human PDAC. To monitor tumor growth, cancer cells that are implanted in mice are often transfected with reporter genes, such as firefly luciferase (Luc), enabling in vivo optical imaging over time. Since Luc can induce an immune response, we aimed to evaluate whether the expression of Luc could affect the growth of KPC tumors in mice by inducing immunogenicity. Although both cell lines, KPC and Luc transduced KPC (KPC-Luc), had the same proliferation rate, KPC-Luc tumors had significantly smaller sizes or were absent 13 days after orthotopic cell implantation, compared to KPC tumors. This coincided with the loss of bioluminescence signal over the tumor region. Immunophenotyping of blood and spleen from KPC-Luc tumor-bearing mice showed a decreased number of macrophages and CD4+ T cells, and an increased accumulation of natural killer (NK) cells in comparison to KPC tumor mice. Higher infiltration of CD8+ T cells was found in KPC-Luc tumors than in their controls. Moreover, the immune response against Luc peptide was stronger in splenocytes from mice implanted with KPC-Luc cells compared to those isolated from KPC wild-type mice, indicating increased immunogenicity elicited by the presence of Luc in the PDAC tumor cells. These results must be considered when evaluating the efficacy of anti-cancer therapies including immunotherapies in immunocompetent PDAC or other cancer mouse models that use Luc as a reporter for bioluminescence imaging.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

Highly Sensitive Analysis of Cervical Mucosal HIV-1 Infection Using Reporter Viruses Expressing Secreted Nanoluciferase.

Ex vivo cervical tissue explant models offer a physiologically relevant approach for studying virus-host interactions that underlie mucosal HIV-1 transmission to women. However, the utility of cervical explant tissue (CET) models has been limited for both practical and technical reasons. These include assay variation, inadequate sensitivity for assessing HIV-1 infection and replication in tissue, and constraints imposed by the requirement for using multiple replica samples of CET to test each experimental variable and assay parameter. Here, we describe an experimental approach that employs secreted nanoluciferase (sNLuc) and current HIV-1 reporter virus technologies to overcome certain limitations of earlier ex vivo CET models. This method augments application of the CET model for investigating important questions involving mucosal HIV-1 transmission.

A Semiquantitative Protein-Fragment Complementation Assay to Study Protein-Protein Interactions of the Polymerase Complex in Cellula.

Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.

Serum Amyloid A3 Promoter-Luciferase Reporter Mice Are Useful for Early Drug-Induced Nephrotoxicity Detection.

Early detection of drug-induced kidney injury is essential for drug development. In this study, multiple low-dose aristolochic acid (AA) and cisplatin (Cis) injections increased renal mRNA levels of inflammation, fibrosis, and renal tubule injury markers. We applied a serum amyloid A3 (Saa3) promoter-driven luciferase reporter (Saa3 promoter-luc mice) to these two tubulointerstitial nephritis models and performed in vivo bioluminescence imaging to monitor early renal pathologies. The bioluminescent signals from renal tissues with AA or CIS injections were stronger than those from normal kidney tissues obtained from normal mice. To verify whether the visualized bioluminescence signal was specifically generated by the injured kidney, we performed in vivo bioluminescence analysis after opening the stomachs of Saa3 promoter-luc mice, and the Saa3-mediated bioluminescent signal was specifically detected in the injured kidney. This study showed that Saa3 promoter activity is a potent non-invasive indicator for the early detection of drug-induced nephrotoxicity.

Bright Red Bioluminescence from Semisynthetic NanoLuc (sNLuc).

Red-shifted bioluminescence is highly desirable for diagnostic and imaging applications. Herein, we report a semisynthetic NanoLuc (sNLuc) based on complementation of a split NLuc (LgBiT) with a synthetic peptide (SmBiT) functionalized with a fluorophore for BRET emission. We observed exceptional BRET ratios with diverse fluorophores, notably in the red (I674/I450 > 14), with a brightness that is sufficient for naked eye detection in blood or through tissues. To exemplify its utility, LgBiT was fused to a miniprotein that binds HER2 (affibody, ZHER2), and the selective detection of HER2+ SK-BR-3 cells over HER2- HeLa cells was demonstrated.

A reproducible and affordable method of conducting luciferase assay.

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection.

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect Schistosoma infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

Exploring the Light-Emitting Agents in Renilla Luciferases by an Effective QM/MM Approach.

Bioluminescence is a fascinating natural phenomenon, wherein organisms produce light through specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc) derived from the sea pansy Renilla reniformis is notable for its blue light emission and has potential applications in bioluminescent tagging. Our study focuses on RLuc8, a variant of RLuc with eight amino acid substitutions. Recent studies have shown that the luminescent emitter coelenteramide can adopt multiple protonation states, which may be influenced by nearby residues at the enzyme's active site, demonstrating a complex interplay between protein structure and bioluminescence. Herein, using the quantum mechanical consistent force field method and the semimacroscopic protein dipole-Langevin dipole method with linear response approximation, we show that the phenolate state of coelenteramide in RLuc8 is the primary light-emitting species in agreement with experimental results. Our calculations also suggest that the proton transfer (PT) from neutral coelenteramide to Asp162 plays a crucial role in the bioluminescence process. Additionally, we reproduced the observed emission maximum for the amide anion in RLuc8-D120A and the pyrazine anion in the presence of a Na+ counterion in RLuc8-D162A, suggesting that these are the primary emitters. Furthermore, our calculations on the neutral emitter in the engineered AncFT-D160A enzyme, structurally akin to RLuc8-D162A but with a considerably blue-shifted emission peak, aligned with the observed data, possibly explaining the variance in emission peaks. Overall, this study demonstrates an effective approach to investigate chromophores' bimolecular states while incorporating the PT process in emission spectra calculations, contributing valuable insights for future studies of PT in photoproteins.

Imaging bioluminescence by detecting localized haemodynamic contrast from photosensitized vasculature.

Bioluminescent probes are widely used to monitor biomedically relevant processes and cellular targets in living animals. However, the absorption and scattering of visible light by tissue drastically limit the depth and resolution of the detection of luminescence. Here we show that bioluminescent sources can be detected with magnetic resonance imaging by leveraging the light-mediated activation of vascular cells expressing a photosensitive bacterial enzyme that causes the conversion of bioluminescent emission into local changes in haemodynamic contrast. In the brains of rats with photosensitized vasculature, we used magnetic resonance imaging to volumetrically map bioluminescent xenografts and cell populations virally transduced to express luciferase. Detecting bioluminescence-induced haemodynamic signals from photosensitized vasculature will extend the applications of bioluminescent probes.