RESUMO
Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.
Assuntos
Diagnóstico por Imagem , Medições Luminescentes , Animais , Luciferases/química , Carbocianinas , Transferência de Energia , Medições Luminescentes/métodos , Mamíferos/metabolismoRESUMO
Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the NIR-BRET templates through the characterization of many coelenterazine (CTZ)-marine luciferase combinations. As a result, we found that NLuc-DBlueC and ALuc47-nCTZ combinations showed luminescence in the blue emission wavelength with excellent BL intensity and stability, for example, the NLuc-DBlueC and ALuc47-nCTZ combinations were 17-fold and 22-fold brighter than their second highest combinations, respectively, and were stably bright in living mammalian cells for at least 10 min. To harness the excellent BL properties to the NIR-BRET systems, NLuc and ALuc47 were genetically fused to fluorescent proteins (FPs), allowing large "blue-to-red" shifts, such as LSSmChe, LSSmKate2, and LSSmNep (where LSS means Large Stokes Shift). The excellent LSSmNep-NLuc combination showed approximately 170 nm large resonance energy shift from blue to red. The established templates were further utilized in the development of new NIR-BRET systems for imaging steroid hormone activities by sandwiching the ligand-binding domain of a nuclear receptor (NR-LBD) between the luciferase and the FP of the template. The NIR-BRET systems showed a specific luminescence signal upon exposure to steroid hormones, such as androgen, estrogen, and cortisol. The present NIR-BRET templates are important additions for utilizing their advantageous imaging of various molecular events with high efficiency and brightness in physiological samples.
Assuntos
Hormônios , Imagem Molecular , Animais , Luciferases/metabolismo , Imagem Molecular/métodos , Esteroides , Medições Luminescentes/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Mamíferos/metabolismoRESUMO
D-Luciferin (D-LH2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH2 and 4',5'-dimethyl-D-LH2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.
Assuntos
Luciferina de Vaga-Lumes , Praguicidas , Luciferases de Vaga-Lume , Luciferinas , Luminescência , Medições Luminescentes/métodosRESUMO
Serum copper levels are biomarkers for copper-related diseases. Quantification of levels of free copper (not bound to proteins) in serum is important for diagnosing Wilson's disease, in which the free copper concentration is elevated. Bioluminescence is commonly used in point-of-care diagnostics, but these assays require genetically engineered luciferase. Here, we developed a luciferase-independent copper detection platform. A luminogenic caged coelenterazine analogue (TPA-H1) was designed and synthesized to detect copper ions in human serum. TPA-H1 was developed by introducing a tris[(2-pyridyl)-methyl]amine (TPA) ligand, which is a Cu+ cleavable caging group, to the carbonyl group at the C-3 position of the imidazopyrazinone scaffold. The luciferin, named HuLumino1, is the product of the cleavage reaction of TPA-H1 with a copper ion and displays "turn-on" bioluminescence signals specifically with human serum albumin, which can be used to quantitatively analyse copper ions. TPA-H1 exhibited a fast cleavage of the protective group, high specificity, and high sensitivity for copper over other metal ions. This novel caged coelenterazine derivative, TPA-H1, can detect free copper ions in serum in a simple "mix-and-read" manner.
Assuntos
Cobre , Imidazóis , Humanos , Luciferases , PirazinasRESUMO
A synthetic luciferin comprising an imidazopyrazinone core, named HuLumino1, was designed to generate specific bioluminescence with human serum albumin (HSA) in real serum samples. HuLumino1 was developed by attaching a methoxy-terminated alkyl chain to C-6 of coelenterazine and by eliminating a benzyl group at C-8. HSA levels were quantified within 5% error margins of an enzyme-linked immunosorbent assay without the need for any sample pretreatments because of the high specificity of HuLumino1.
Assuntos
Imidazóis/química , Luminescência , Pirazinas/química , Albumina Sérica Humana/metabolismo , Alquilação , HumanosRESUMO
There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 µM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.
Assuntos
Imidazóis/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Molecular/métodos , Pirazinas/química , Compostos de Sulfidrila/química , Animais , Células COS , Chlorocebus aethiops , Estrutura Molecular , Imagem Óptica/métodos , SensilasRESUMO
A coelenterazine (CTZ) analogue emitting near-infrared (NIR) bioluminescence was synthesized for through-bond energy transfer (TBET)-based imaging modalities. The analogue, named Cy5-CTZ, was prepared by conjugating cyanine-5 (Cy5) dye to CTZ through an acetylene linker. This novel derivative is intrinsically fluorescent and emits NIR-shifted luminescence upon reacting with an appropriate luciferase, the Renilla luciferase. This Cy5-CTZ substrate is optically stable in physiological samples and rapidly permeabilize through the plasma membrane into the cytosolic compartment of live cells.
Assuntos
Carbocianinas/química , Imidazóis/química , Substâncias Luminescentes/química , Medições Luminescentes , Pirazinas/química , Transferência de Energia , Raios Infravermelhos , Estrutura MolecularRESUMO
Native coelenterazine (nCTZ) is a common substrate to most marine luciferases and photoproteins. In this study, nine novel dye- and azide-conjugated CTZ analogues were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of the nCTZ backbone to obtain bulkiness-driven substrate specificity and potential chemiluminescence/bioluminescence resonance energy transfer (C/BRET). The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly biased bioluminescence to ALucs and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living animal cells or lysates. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than nCTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). No or less spectral overlap occurs among [Furimazine-NanoLuc], [6-N3-CTZ-ALuc26], [6-pi-OH-CTZ-RLuc8.6], and [6-N3-CTZ-RLuc8.6] pairs, because of the substrate-driven luciferase specificity and color shifts, providing a crosstalk-free multiplex bioassay platform. The unique bioluminescence system appends a new toolbox to bioassays and multiplex molecular imaging platforms. This study is the first example that systematically synthesized fluorescent dye-conjugated CTZs and applied them for a bioluminescence assay system.
Assuntos
Azidas/química , Corantes Fluorescentes/química , Imidazóis/química , Pirazinas/química , Animais , Azidas/síntese química , Células COS , Chlorocebus aethiops , Corantes Fluorescentes/síntese química , Imidazóis/síntese química , Luciferases/química , Substâncias Luminescentes/química , Medições Luminescentes , Imagem Molecular , Pirazinas/síntese químicaRESUMO
Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.
Assuntos
Luciferases de Renilla/metabolismo , Sondas Moleculares/metabolismo , Mapeamento de Interação de Proteínas/métodos , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biomecânicos , Células COS , Chlorocebus aethiops , Humanos , Luciferases de Renilla/química , Medições Luminescentes/métodos , Sondas Moleculares/química , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Renilla/química , Renilla/enzimologia , Serina-Treonina Quinases TOR/química , Proteínas de Ligação a Tacrolimo/químicaRESUMO
Enzymatic reactions that involve a luminescent substrate (luciferin) and enzyme (luciferase) from luminous organisms enable a luminescence detection of target proteins and cells with high specificity, albeit that conventional assay design requires a prelabeling of target molecules with luciferase. Here, we report a luciferase-independent luminescence assay in which the target protein directly catalyzes the oxidative luminescence reaction of luciferin. The SARS-CoV-2 antigen (spike) protein catalyzes the light emission of Cypridina luciferin, whereas no such catalytic function was observed for salivary proteins. This selective luminescence reaction is due to the enzymatic recognition of the 3-(1-guanidino)propyl group in luciferin at the interfaces between the units of the spike protein, allowing a specific detection of the spike protein in human saliva without sample pretreatment. This method offers a novel platform to detect virus antigens simply and rapidly without genetic manipulation or antibodies.
RESUMO
Asymmetric hydrogenation of α-alkyl-substituted ß-keto esters and amides with the DIPSkewphos/3-AMIQ-Ru(II) catalyst system through dynamic kinetic resolution was examined. A series of ß-keto esters and amides with a simple or functionalized α-alkyl group were applicable to this reaction, affording the α-substituted ß-hydroxy esters and amides in ≥99% ee (anti/syn ≥ 99:1) in many cases. The 5 g scale reaction was readily achieved. The mode of enantio- and diastereoselection in the transition state model was proposed.
RESUMO
The enzymatic luminescence reactions of fireflies are accelerated in the presence of biomolecular condensates comprising a positively charged peptide and ATP. We revealed that this acceleration is caused by the enrichment of reaction elements, local pH changes, and promotion of inhibitory intermediate dissociation, improving the bioluminescence quantum yield by approximately 10%.
Assuntos
Vaga-Lumes , Luciferases de Vaga-Lume , Animais , Vaga-Lumes/química , Condensados Biomoleculares , LuminescênciaRESUMO
Coelenterazine (CTZ) is the most general substrate for marine luciferases. The present protocol introduces a near-infrared (NIR ) bioluminescence (BL) imaging of mammalian cells with a cyanine-5 (Cy5) dye-conjugated CTZ . This unique Cy5-conjugated CTZ, named Cy5-CTZ , can act as a dual optical readout emitting both fluorescence (FL) and BL. The Cy5-CTZ exerts through-bond energy transfer (TBET)-based imaging modalities for mammalian cells. This novel derivative, Cy5-CTZ , is intrinsically fluorescent and emits NIR-shifted BL when reacting with an appropriate luciferase , such as Renilla luciferase (RLuc). The protocol exemplifies a unique live-cell imaging with Cy5-CTZ that is optically stable in physiological samples and rapidly permeabilize through plasma membrane and emit NIR-BL in live mammalian cells.
Assuntos
Carbocianinas/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Molecular/métodos , Pirazinas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Células COS , Chlorocebus aethiops , Transferência de EnergiaRESUMO
Coelenterazine (CTZ) is a common substrate to most marine luciferases and photoproteins. The present protocol introduces mammalian cell imaging with nine novel dye- and azide-conjugated CTZ analogues, which were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of CTZ backbone. The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly selective bioluminescence (BL) to artificial luciferases (ALucs) and ca. 130 nm blue-shifted BL with Renilla luciferase variant 8.6 (RLuc8.6) in mammalian cells. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than CTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). The present protocol shows that the minimal spectral overlap occurs among the pairs of [Furimazine/NanoLuc], [6-N3-CTZ/ALuc26], [6-pi-OH-CTZ/RLuc8.6], and [6-N3-CTZ/RLuc8.6] because of the substrate-driven luciferase specificity or color shifts, convincing a cross talk-free multiplex bioassay platform. The present protocol introduces a new toolbox to bioassays and multiplex molecular imaging platforms for mammalian cells.
Assuntos
Azidas/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Molecular/métodos , Pirazinas/química , Animais , Células COS , Chlorocebus aethiopsRESUMO
Spectral overlaps in fluorescence (FL) and bioluminescence (BL) commonly cause optical cross talks. The present protocol introduces five different lineages of coelenterazine (CTZ) analogues, which have selectivity to a specific luciferase, and thus cross talk-free. For example, some CTZ analogues with ethynyl or styryl groups display dramatically biased BL to specific luciferases and pH by modifying the functional groups at the C-2 and C-6 positions of the imidazopyridinne backbone of CTZ. The optical cross talk-free feature is exemplified with the multiplex system, which simultaneously illuminated antiestrogenic and rapamycin activities without optical cross talks. This unique protocol contributes to specific and high-throughput BL imaging of multiple optical readouts in mammalian cells without optical contamination.
Assuntos
Alcinos/química , Imidazóis/química , Luciferases/metabolismo , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Estirenos/química , Animais , Células COS , Chlorocebus aethiops , Moduladores de Receptor Estrogênico/farmacologia , Imunossupressores/farmacologia , Luciferases/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
The present protocol demonstrates a novel mammalian cell imaging platform exerting a bioluminescence resonance energy transfer (BRET) system. This platform achieves a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) with the development of a unique coelenterazine (CTZ) derivative named BBlue2.3 and a fusion reporter protein probe named iRFP-RLuc8.6-535SG. The best NIR-BRET shift was achieved by tuning the blue emission peak of BBlue2.3 to a Soret band of the iRFP. In mammalian cells, BBlue2.3 emits light that is ~50-fold brighter than DeepBlueC in cell imaging when combined with RLuc8.6-535SG. This NIR-BRET platform is sufficiently brighter to be used for imaging live mammalian cells at single-cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. This unique optical platform provides the brightest NIR-BLI template that can be used for imaging a diverse group of cellular events in living subjects.
Assuntos
Neoplasias da Mama/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Imidazóis/química , Luciferases/metabolismo , Medições Luminescentes/métodos , Imagem Óptica/métodos , Pirazinas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Apoptose , Neoplasias da Mama/metabolismo , Proliferação de Células , Feminino , Humanos , Substâncias Luminescentes/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As protein-protein interactions (PPI) have been mostly investigated in cellulo or in vivo, it is unclear whether the PPI-based imaging schemes are practically valid in a bioanalytical means in vitro. The present study exemplifies the PPI in vitro inside a unique single-chain probe, named TP2.4, which carries a full-length artificial luciferase (ALuc) sandwiched in between two model proteins of interest, e.g., FKBP and FRB, expressed in E. coli, and purified. We found that the TP2.4 efficiently recognizes its ligand in vitro and varies its molecular kinetics: i.e., rapamycin boosts the enzymatic affinities (Km) of TP2.4 to its substrates, but does not or only weakly influences the turnover rates (Kcat) and the maximal velocity (Vmax). The corresponding circular dichroism (CD) study shows that rapamycin weakly contributes to the enhancement of the α-helical contents in TP2.4. Kinetic constants according to the substrates revealed that a coelenterazine derivative, 6-N3-CTZ, exerted the best catalytic efficiency and the greatest variance in the total photon counts. The present study is the first in vitro example that demonstrates how intramolecular PPI works in a purified single-chain bioluminescent probe and what factors practically influence the biochemistry.
Assuntos
Sondas Moleculares , Mapeamento de Interação de Proteínas/métodos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Dicroísmo Circular , Cinética , Luciferases/genética , Medições Luminescentes , Plasmídeos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina-Treonina Quinases TOR/genética , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Retinoic acid (RA) is a key metabolite necessary for embryonic development and differentiation in vertebrates. We demonstrate the utility of genetically encoded, ligand-activatable single-chain bioluminescence probes for detecting RAs from different biological sources. We examined 13 different molecular designs to identify an efficient single-chain probe that can quantify RA with significant sensitivity. The optimal probe consisted of four components: the N- and C-terminal fragments of artificial luciferase variant-16 (ALuc16), the ligand binding domain of retinoic acid receptor α (RARα LBD), and an LXXLL interaction motif. This probe showed a 5.2-fold greater bioluminescence intensity in response to RA when compared to the vehicle control in live mammalian cells. The probe was highly selective to all-trans-RA (at-RA), and highly sensitive in determining at-RA levels in cells derived from tumor xenografts created using MDA-MB-231 cells engineered to stably express the probe. We also detected RA levels in serum and cerebrospinal fluid. Using this probe, the detection limit for at-RA was â¼10-9.5 M, with a linear range of two orders. We present a highly useful technique to quantitatively image endogenous at-RA levels in live mammalian cells expressing novel single-chain bioluminescence probes.
Assuntos
Corantes Fluorescentes/química , Tretinoína/análise , Animais , Sítios de Ligação , Linhagem Celular , Feminino , Humanos , Ligantes , Camundongos Endogâmicos BALB C , Imagem Óptica , Receptor alfa de Ácido Retinoico/química , Receptor alfa de Ácido Retinoico/metabolismo , Imagem Individual de Molécula , Tretinoína/metabolismoRESUMO
Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named "Bottle Blue (BBlue)" and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including proteinâprotein interactions and cancer metastasis.