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Schematic of a self-contained Lux operon system. The lux operon usually contains five different genes: luxC, luxD, luxA, luxB, and luxE. These five genes constitute the wildtype version of lux operon that can be found in Photorhabdus luminescens; this figure shows the modified version of lux operon that contains an additional frp gene for better operational efficiency when expressed in certain organisms. luxA and luxB code two subunits for dimeric LuxAB luciferase; luxC, luxD, and luxE code relative enzymes, which can form a dodecamer protein complex to produce fatty aldehyde substrate for LuxAB; and frp gene codes the Flavin reductase P that can be used to shift the natural cellular balance between FMN and FMNH 2 . In the presence of oxygen, LuxAB oxidizes fatty aldehyde and FMNH 2 to generate cyan luminescence (λ = 490 nm). The oxidized products, fatty acid and FMN, can be recycled to re-form substrates. The continuous run of Lux system only requires the supplies of ATP, NADPH, and oxygen, so it is self-contained and able to constantly produce luminescence as long as the host cell is alive.
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Proteins are the elementary machinery of life, and their functions are carried out mostly by molecular interactions. Among those interactions, protein–protein interactions (PPIs) are the most important as they participate in or mediate all essential biological processes. However, many common methods for PPI investigations are slightly unreliable an...
Citations
... The expected emission peaks at 469 nm and 468 nm for GLuc and NLuc, respectively ( Figure 2B), were observed. However, the total bioluminescence for GLuc was found to be~10 4 counts per second (CPS) (Figure 2B, inset; left panel), while the total bioluminescence for NLuc was found to bẽ 10 7 CPS ( Figure 2B, inset; right panel) (Sun et al., 2016;Biswas et al., 2008). Based on the higher bioluminescence, we selected NLuc as the BRET donor for assembling the BioODs. ...
The need for the early detection of emerging pathogenic viruses and their newer variants has driven the urgent demand for developing point-of-care diagnostic tools. Although nucleic acid-based methods such as reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and loop-mediated isothermal amplification (LAMP) have been developed, a more facile and robust platform is still required. To address this need, as a proof-of-principle study, we engineered a prototype—the versatile, sensitive, rapid, and cost-effective bioluminescence resonance energy transfer (BRET)-based biosensor for oligonucleotide detection (BioOD). Specifically, we designed BioODs against the SARS-CoV-2 parental (Wuhan strain) and B.1.617.2 Delta variant through the conjugation of specific, fluorescently modified molecular beacons (sensor module) through a complementary oligonucleotide handle DNA functionalized with the NanoLuc (NLuc) luciferase protein such that the dissolution of the molecular beacon loop upon the binding of the viral oligonucleotide will result in a decrease in BRET efficiency and, thus, a change in the bioluminescence spectra. Following the assembly of the BioODs, we determined their kinetics response, affinity for variant-specific oligonucleotides, and specificity, and found them to be rapid and highly specific. Furthermore, the decrease in BRET efficiency of the BioODs in the presence of viral oligonucleotides can be detected as a change in color in cell phone camera images. We envisage that the BioODs developed here will find application in detecting viral infections with variant specificity in a point-of-care-testing format, thus aiding in large-scale viral infection surveillance.
... Both FRET and BRET have been widely used to study PPIs in various cellular contexts, including signal transduction pathways, protein trafficking, and protein-protein interactions involved in disease pathways [116]. In recent years, improvements in the design and sensitivity of both FRET and BRET sensors have enabled more accurate and quantitative measurement of PPIs in living cells [117,118]. ...
Protein-protein interactions (PPIs) regulate crucial physiological and pathological processes. PPIs are considered a class of biological targets almost infeasible for small molecules because the binding surfaces are usually large and shallow. Peptides are molecules able to bind to these drug targets; they can be used as modulators and mimic one of the interaction partners. This review details the advances in in silico peptide design and experimental approaches for the evaluation of PPI-based peptides.
... However, the reaction catalyzed by Fluc needs ATP and cofactor magnesium, which is different from CTZ-consuming luciferases. Along with its big size of 62 kDa and sensitivity to temperature and ionic strength, Fluc is not widely used in BRET like CTZ-consuming luciferases [37]. Bacterial luciferase from Photorhabdus luminescens has distinctive features. ...
Resonance energy transfer technologies have achieved great success in the field of analysis. Particularly, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) provide strategies to design tools for sensing molecules and monitoring biological processes, which promote the development of biosensors. Here, we provide an overview of recent progress on FRET- and BRET-based biosensors and their roles in biomedicine, environmental applications, and synthetic biology. This review highlights FRET- and BRET-based biosensors and gives examples of their applications with their design strategies. The limitations of their applications and the future directions of their development are also discussed.
... As reported, nanoluciferase (Nluc), the smallest commercially available luciferase reporter (19 kDa), can offer the brightest bioluminescence with over a 150-fold enhancement compared with that of traditional luciferase [26,27]. Nluc has been used as an excellent fusion protein partner to develop bifunctional tracers for antibody screening [26], protein-protein interaction analysis [28], disease diagnosis [29], bioluminescence imaging [30] and immunoassay [31,32]. ...
Ochratoxin A (OTA), one of the best-known mycotoxins, causes problems concerning food safety with potential toxic effects in humans and animals. So, it is crucial to develop simple and sensitive methods for the detection of OTA. Herein, a nanoluciferase–nanobody fusion protein (Nb28-Nluc)-retaining antibody recognition and enzymatic activity was first prepared, which was then applied as a bifunctional tracer to construct a one-step bioluminescent enzyme-linked immunosorbent assay (BLEIA) for OTA in coffee samples. On the basis of Nb28-Nluc, the BLEIA can be completed with a one-step incubation and detection, with only a substrate replacement from 3,3′,5,5′-tetramethylbenzidine (TMB) to a Nluc assay reagent (Furimazine). Under the optimal experimental conditions, the proposed one-step BLEIA achieved a detection limit of 3.7 ng/mL (IC10) within 3 h. Moreover, the BLEIA method showed good repeatability and accuracy in the spike recovery experiments with recoveries of 83.88% to 120.23% and relative standard deviations (RSDs) of 5.2% to 24.7%, respectively. Particularly, the BLEIA displayed superior performances, such as fewer operations and more rapid and sensitive detection as compared with Nb28-based enzyme-linked immunosorbent assay. Therefore, the proposed one-step BLEIA has great potential for the sensitive and accurate screening of OTA in food samples.
... BRET ratio = (YFP emission)/(RLuc emission) BRET pair − (YFP emission)/(RLuc emission) BRET donor For BRET saturation experiments, HEK293T cells were transfected with appropriate amounts of RLuc and YFP expression plasmids, ensuring similar levels of RLuc expression. BRET saturation curves were generated using the GraphPad Prism software (Graphpad Software Inc., San Diego, California, USA) by plotting each individual BRET ratio value to the YFPnet/RLuc signal, and interpolating such values using the one-site binding hyperbola function of GraphPad Prism (Graphpad Software Inc.) to calculate BRET max (B max ) and BRET 50 (B 50 ) values, indicative of maximum energy transfer and relative affinity of each BRET pair tested, respectively [39]. For BRET inhibition experiments, cells were transfected with appropriate BRET plasmids to generate a BRET signal similar to 50% of the BRET max value, in the presence of increasing concentrations of either pDESTnFLAG-UL44 or pDESTnFLAG-UL42, and the BRET ratio values were plotted against the concentration of pDESTnFLAG plasmid used for transfection. ...
... Our data clearly indicate that BRET assays allow to easily monitor the interaction of biologically relevant protein-protein interactions crucial for HCMV genome replication, and they could potentially be used to screen compounds inhibiting such interactions and thus viral replication. Since previous studies implied that expression dynamics of BRET donor and acceptor proteins could influence the success of such approaches in drug discovery [39], we aimed at comparing three different expression systems for BRET pairs: (i) the most simple expression system relies on transient expression of both BRET donor and acceptor proteins, and corresponds to the approach already described in Figures 3 and 4; other expression systems proposed rely either on (ii) stable expression of the BRET acceptor (YFP-fusion protein) and transient expression of the BRET donor (RLuc-fusion protein) or on (iii) stable expression of both BRET pairs. Therefore, we set out to generate YFP-UL44 stably expressing cells. ...
Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.
... Both for FRET and BiFC, a fluorescent protein could be replaced by a luciferase enzyme. Since no excitation light is needed here, problems such as photo-toxicity, autofluorescence, photobleaching and crossexcitation are avoided to a large extent (Sun et al. 2016). BiFC analysis could be performed using a luciferase enzyme instead of a fluorescent protein, in this case the assay is called a splitluciferase system (Fukutani et al. 2017). ...
Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements.
... All these properties of NanoLuc have led to its utility in a variety of applications including resonance energy transfer-based protein-protein interaction determination (BRET), gene regulation, and monitoring protein stability in diseased conditions [32][33][34][35][36][37][38][39]. In fact, a number of biosensing strategies have been devised based on the BRET phenomenon [40][41][42][43][44][45][46][47][48]. ...
Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.
... Fluorescent proteins are most often used as acceptors in BRET [122]. The proteins of interest are genetically fused either with the donor or the acceptor [123][124][125][126]. Qdots can also be used as acceptors in BRET since the donor is not excited by light absorption and directexcitation of the acceptor is therefore not an issue [109,112]. ...
Although the theoretical foundations of Förster resonance energy transfer (FRET) were laid in the 1940s as part of the quantum physical revolution of the 20th century, it was only in the 1970s that it made its way to biology as a result of the availability of suitable measuring and labeling technologies. Thanks to its ease of application, FRET became widely used for studying molecular associations on the nanometer scale. The development of superresolution techniques at the turn of the millennium promised an unprecedented insight into the structure and function of molecular complexes. Without downplaying the significance of superresolution microscopies this review expresses our view that FRET is still a legitimate tool in the armamentarium of biologists for studying molecular associations since it offers distinct advantages and overcomes certain limitations of superresolution approaches.
... In vivo, the yeast or mammalian two/three hybridization system has been widely applied to identify novel PPIs [8,16]. For the transient PPIs, many developed approaches such as FRET, BRET and BiFC were applied to confirm the PPIs [12,[17][18][19][20][21]. To capture the novel PPI in vivo, the proximity-dependent labeling (PDL) technologies were also developed that can be applied to analyze both stable and transient PPIs [22]. ...
The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.
... BRET is a naturally occurring phenomenon for the efficient spectral conversion of short-wavelength luminescence into a longer wavelength, which can be used in the real-time monitoring of PPIs in living cells, cell extracts, or purified preparations (Sun et al., 2016). The BRET approach resembles FRET in every way, except that a bioluminescent energy donor, often Renilla luciferase (Rluc), replaces the fluorescent donor (Subramanian et al., 2006). ...
... The fluorescent energy acceptor, for example, YFP, is excited by energy released from the oxidation of luciferin by luciferase, and thus emits fluorescence. In the presence of luciferin but no protein interaction, the luciferase will con-stantly emit light while the YFP will not, indicating that there is no direct interaction between the proteins of interest (Sun et al., 2016) (Figure 1B). On the other hand, if the excited electron energy of luciferase (donor) is transferred to a fluorescent protein (acceptor), the light is emitted at a longer wavelength, indicating that the proteins of interest are likely to be interacting with each other. ...
... The absence of external excitation means that the use of BRET avoids problems such as photo-toxicity, photobleaching of light-sensitive molecules (Xie et al., 2011), and inhomogeneous excitation due to light scattering (Boute et al., 2002). As a result, BRET has almost no background luminescence or false signals generated by unintentional excitations with external light, meaning the signal-to-noise ratio of this technique is much higher than that of FRET and thus the sensitivity and accuracy of the measurements are increased (Sun et al., 2016). Many types of luciferases and fluorescent proteins exist, and each combination used for BRET may have certain advantages over the others. ...
Detecting protein-protein interactions (PPIs) provides fundamental information for understanding biochemical processes such as the transduction of signals from one cellular location to another; however, traditional biochemical techniques cannot provide sufficient spatio-temporal information to elucidate these molecular interactions in living cells. Over the past decade, several new techniques have enabled the identification and characterization of PPIs. In this review, we summarize three main techniques for detecting PPIs in vivo, focusing on their basic principles and applications in biological studies. We place a special emphasis on their advantages and limitations, and, in particular, we introduced some uncommon new techniques, such as single-molecule FRET (smFRET), FRET-fluorescence lifetime imaging microscopy (FRET-FLIM), cytoskeleton-based assay for protein-protein interaction (CAPPI) and single-molecule protein proximity index (smPPI), highlighting recent improvements to the established techniques. We hope that this review will provide a valuable reference to enable researchers to select the most appropriate technique for detecting PPIs.
