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Molecular and Cellular Biology Feb 1987The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six...
The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
Topics: Animals; Base Sequence; Cell Line; Chlorocebus aethiops; Cloning, Molecular; Coleoptera; DNA; Firefly Luciferin; Gene Expression Regulation; Genes; Luciferases; Promoter Regions, Genetic; RNA, Messenger
PubMed: 3821727
DOI: 10.1128/mcb.7.2.725-737.1987 -
Bioconjugate Chemistry May 2016The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical... (Review)
Review
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.
Topics: Animals; Biomedical Research; Humans; Luciferases; Luminescence; Nanostructures; Nanotechnology
PubMed: 27045664
DOI: 10.1021/acs.bioconjchem.6b00112 -
Nature Methods Aug 2020Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange...
Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
Topics: Animals; Enzyme Assays; Furans; Luciferases; Luminescent Measurements; Luminescent Proteins; Substrate Specificity
PubMed: 32661427
DOI: 10.1038/s41592-020-0889-6 -
Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chemical Biology Nov 2012Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have...
Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
Topics: Animals; Cell Line; Crustacea; Enzyme Stability; Fireflies; Gene Expression; Genes, Reporter; Humans; Luciferases; Luminescent Agents; Models, Molecular; Protein Engineering; Pyrazines; Recombinant Fusion Proteins; Renilla; Temperature
PubMed: 22894855
DOI: 10.1021/cb3002478 -
Current Opinion in Chemical Biology Aug 2021Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a... (Review)
Review
Bioluminescence (BL) relies on the enzymatic reaction between luciferase, a substrate conventionally named luciferin, and various cofactors. BL imaging has become a widely used technique to interrogate gene expression and cell fate, both in small and large animal models of research. Recent developments include the generation of improved luciferase-luciferin systems for deeper and more sensitive imaging as well as new caged luciferins to report on enzymatic activity and other intracellular functions. Here, we critically evaluate the emerging tools for BL imaging aiming to provide the reader with an updated compendium of the latest developments (2018-2020) and their notable applications.
Topics: Animals; Blood-Brain Barrier; Cell Line; Cell Membrane Permeability; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Expression; Gene Knock-In Techniques; Humans; Luciferases; Luminescent Agents; Luminescent Measurements; Optical Imaging; Structure-Activity Relationship
PubMed: 33770744
DOI: 10.1016/j.cbpa.2021.02.005 -
Biosensors Oct 2022A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an...
A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an ATP sensor that is low-cost and easy to use. Gold nanoparticles provide excellent biocompatibility for enzyme immobilization. We investigated the effect of luciferase proximity with citrate-coated gold, silver, and gold-silver core-shell nanoparticles, gold nanorods, and BSA-Au nanoclusters. The effect of metal nanoparticles on the activity of luciferases was recorded by the luminescence assay, which was 3-5 times higher than free enzyme. The results showed that the signal stability in presence of nanoparticles improved and was reliable up to 6 h for analytes measurements. It has been suggested that energy is mutually transferred from luciferase bioluminescence spectra to metal nanoparticle surface plasmons. In addition, we herein report the 27-base DNA aptamer for adenosine-5'-triphosphate (ATP) as a suitable probe for the ATP biosensor based on firefly luciferase activity and AuNPs. Due to ATP application in the firefly luciferase reaction, the increase in luciferase activity and improved detection limits may indicate more stability or accessibility of ATP in the presence of nanoparticles. The bioluminescence intensity increased with the ATP concentration up to 600 µM with a detection limit of 5 µM for ATP.
Topics: Gold; Metal Nanoparticles; Silver; Luciferases, Firefly; Adenosine Triphosphate; Luciferases
PubMed: 36354427
DOI: 10.3390/bios12110918 -
Scientific Reports Jul 2022Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as...
Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.
Topics: Animals; Fireflies; Genes, Reporter; Indicators and Reagents; Luciferases; Luciferases, Firefly; Luminescence
PubMed: 35798760
DOI: 10.1038/s41598-022-15624-6 -
Biochemistry. Biokhimiia Jun 2015Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish.... (Review)
Review
Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.
Topics: Animals; Imidazoles; Luciferases; Luminescent Proteins; Pyrazines
PubMed: 26531017
DOI: 10.1134/S0006297915060073 -
Expert Opinion on Drug Discovery Jan 2023Luminescence-based technologies, specifically bioluminescence and chemiluminescence, are powerful tools with extensive use in drug discovery. Production of light during... (Review)
Review
INTRODUCTION
Luminescence-based technologies, specifically bioluminescence and chemiluminescence, are powerful tools with extensive use in drug discovery. Production of light during chemiluminescence and bioluminescence, unlike fluorescence, doesn't require an excitation light source, resulting in high signal-to-noise ratio, less background interference, and no issues from phototoxicity and photobleaching. These characteristics of luminescence technologies offer unique advantages for experimental designs, allowing for greater flexibility to target a wide range of proteins and biological processes for drug discovery at different stages.
AREAS COVERED
This review provides a basic overview of luciferase-based technologies and details recent advances and use cases of luciferase and luciferin variations and their applicability in the drug discovery toolset. The authors expand upon specific applications of luciferase technologies, including chemiluminescent and bioluminescent-based microscopy. Finally, the authors lay out forward-looking statements on the field of luminescence and how it may shape the translational scientists' work moving forward.
EXPERT OPINION
The demand for improved luciferase and luciferin pairs correlates strongly with efforts to improve the sensitivity and robustness of high-throughput assays. As luminescent reporter systems improve, so will the expansion of use cases for luminescence-based technologies in early-stage drug discovery. With the synthesis of novel, non-enzymatic chemiluminescence-based probes, which previously were restrained to only basic research applications, they may now be readily implemented in drug discovery campaigns.
Topics: Humans; Luminescence; Luciferases; Technology; Drug Discovery
PubMed: 36562206
DOI: 10.1080/17460441.2023.2160441 -
Biosensors Feb 2023Antibody measurements play a central role in the diagnosis of many autoimmune and infectious diseases. One antibody detection technology, Luciferase Immunoprecipitation... (Review)
Review
Antibody measurements play a central role in the diagnosis of many autoimmune and infectious diseases. One antibody detection technology, Luciferase Immunoprecipitation Systems (LIPS), utilizes genetically encoded recombinant luciferase antigen fusion proteins in an immunoglobulin capture format to generate robust antibody measurement with high diagnostic sensitivity and specificity. The LIPS technology has been highly useful in detecting antibodies for research diagnostics and the discovery of new autoantigens. The methodology of the assay requires immunoglobulin binding reagents such as protein A/G beads and washing steps to process the immune complex before antibody levels are measured by light production with a luminometer. Recently, simplified mix and read immunoassays based on split components of the nanoluciferase enzyme in a complementation format have been developed for antibody measurements without requiring immunoglobulin-capturing beads or washing steps. The mix and read immunoassays utilize two or three nanoluciferase fragments which when reconstituted via antigen-specific antibody binding generate a functional enzyme. At present, these split luciferase tests have been developed mainly for detecting SARS-CoV-2 antibodies. Here, we describe the traditional LIPS technology and compare it to the new split luciferase methodologies focusing on their technical features, strengths, limitations, and future opportunities for diagnostic research, and clinical applications.
Topics: Humans; COVID-19; SARS-CoV-2; Luciferases; Immunoassay; Antibodies, Viral
PubMed: 36979515
DOI: 10.3390/bios13030303