RESUMO
Bioluminescence imaging with luciferase-luciferin pairs is commonly used for monitoring biological processes in cells and whole organisms. Traditional bioluminescent probes are limited in scope, though, as they cannot be easily distinguished in biological environments, precluding efforts to visualize multicellular processes. Additionally, many luciferase-luciferin pairs emit light that is poorly tissue penetrant, hindering efforts to visualize targets in deep tissues. To address these issues, we synthesized a set of π-extended luciferins that were predicted to be red-shifted luminophores. The scaffolds were designed to be rotationally labile such that they produced light only when paired with luciferases capable of enforcing planarity. A luciferin comprising an intramolecular "lock" was identified as a viable light-emitting probe. Native luciferases were unable to efficiently process the analog, but a complementary luciferase was identified via Rosetta-guided enzyme design. The unique enzyme-substrate pair is red-shifted compared to well-known bioluminescent tools. The probe set is also orthogonal to other luciferase-luciferin probes and can be used for multicomponent imaging. Four substrate-resolved luciferases were imaged in a single session. Collectively, this work provides the first example of Rosetta-guided design in engineering bioluminescent tools and expands the scope of orthogonal imaging probes.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/química , Substâncias Luminescentes/química , Medições Luminescentes , Luciferina de Vaga-Lumes/síntese química , Luciferases/metabolismo , Luminescência , Substâncias Luminescentes/síntese química , Estrutura MolecularRESUMO
d-Luciferin is a popular bioluminescent substrate of luciferase in the presence of ATP. It is used in luciferase-based bioluminescence imaging and cell-based high-throughput screening applications. Herein, the iodination of d-luciferin was undertaken and explored as a bioluminescence probe without the need for light excitation to sensitively trace and image carbon monoxide (CO) in liver cancer cells. The bioluminescent probe (7'-iodo-luciferin) exhibited excellent selectivity for CO detection in vitro. This new probe could image exogenous and endogenous CO in the luciferase-transfected cancer cells. This new probe might be used for evaluating the roles of CO in various biological processes.
Assuntos
Monóxido de Carbono/análise , Luciferina de Vaga-Lumes/análogos & derivados , Substâncias Luminescentes/química , Linhagem Celular Tumoral , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/toxicidade , Células HEK293 , Halogenação , Humanos , Limite de Detecção , Luciferases de Vaga-Lume/química , Luminescência , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/toxicidade , Medições Luminescentes/métodos , Compostos Organometálicos/químicaRESUMO
Iron plays an essential role in biological system. An approach for in vivo imaging of this metal ion is needed to investigate its complex contributions to physiological and pathological processes. Herein, we present a bioluminescent probe FP-1 as a powerful tool for targeting Fe2+ detection in vitro and in vivo. The turn-on sensing scheme is based on the caged strategy of luciferin-luciferase system. FP-1 not only can detect accumulations of exogenous Fe2+ in living animal, but also has the capability of monitoring labile endogenous Fe2+ levels in animal model of sepsis. Implementation of this technique provides a valuable opportunity for understanding underlying mechanisms of Fe2+ in biological processes and disease states.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Ferro/análise , Substâncias Luminescentes/química , Piridinas/química , Sepse/metabolismo , Animais , Modelos Animais de Doenças , Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/síntese química , Ferro/metabolismo , Limite de Detecção , Luciferases de Vaga-Lume/química , Substâncias Luminescentes/síntese química , Medições Luminescentes/métodos , Masculino , Camundongos Transgênicos , Piridinas/síntese químicaRESUMO
An original gram-scale synthesis of O-acetylated forms of coelenterazine, furimazine or hydroxy-bearing analogues of luciferins is described. The comparison over two hours of their bioluminescence, using the nanoKAZ/NanoLuc luciferase, provides remarkable insights useful for the selection of a substrate adapted for a given application.
Assuntos
Luciferina de Vaga-Lumes/síntese química , Imidazóis/síntese química , Pirazinas/síntese química , Acetilação , Animais , Vaga-Lumes , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/química , Imidazóis/química , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Estrutura Molecular , Pirazinas/químicaRESUMO
Bioorthogonal chemistry has developed significant over the past few decades, to the particular benefit of molecular imaging. Bioluminescence imaging (BLI) along with other imaging modalities have significantly benefitted from this chemistry. Here, we review bioorthogonal reactions that have been used to signific antly broaden the application range of BLI.
Assuntos
Técnicas de Química Sintética , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Luciferases/metabolismo , Imagem Molecular/métodos , Animais , Luciferina de Vaga-Lumes/análogos & derivados , Genes Reporter , Humanos , Luciferases/genética , Medições Luminescentes , OxirreduçãoRESUMO
Mercury is a highly toxic environmental pollutant that negatively affects human health. Thus, an in vivo method for noninvasive imaging of mercury(ii) and visualization of its accumulation within living systems would be advantageous. Herein, we describe a reaction-based bioluminescent probe for detection of mercury(ii) in vitro and accumulation in vivo. The application of this probe would help to shed light on the intricate contributions of mercury(ii) to various physiological and pathological processes.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Substâncias Luminescentes/química , Mercúrio/metabolismo , Animais , Linhagem Celular Tumoral , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/toxicidade , Humanos , Luciferases/química , Luminescência , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/toxicidade , Medições Luminescentes/métodos , Camundongos , Distribuição TecidualRESUMO
Compared to the broad palette of fluorescent molecules, there are relatively few structures that are competent to support bioluminescence. Here, we focus on recent advances in the development of luminogenic substrates for firefly luciferase. The scope of this light-emitting chemistry has been found to extend well beyond the natural substrate and to include enzymes incapable of luciferase activity with d-luciferin. The broadening range of luciferin analogues and evolving insight into the bioluminescent reaction offer new opportunities for the construction of powerful optical reporters of use in live cells and animals.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Luciferases de Vaga-Lume/química , Amidoidrolases/química , Animais , Coenzima A Ligases/química , Luciferina de Vaga-Lumes/síntese química , Humanos , Luminescência , Estrutura MolecularRESUMO
As a kind of biologically important reactive oxygen species (ROS), hypochlorite (ClO-) plays a crucial role in many physiological processes. As such, endogenous ClO- is a powerful antibacterial agent during pathogen invasion. Nonetheless, excessive endogenous ClO- could pose a health threat to mammalian animals including humans. However, the detection of endogenous ClO- by bioluminescence probes in vivo remains a considerable challenge. Herein, based on a caged strategy, we developed a turn-on bioluminescent probe 1 for the highly selective detection of ClO-in vitro and imaging endogenous ClO- in a mouse inflammation model. We anticipate that such a probe could help us understand the role of endogenous ClO- in a variety of physiological and pathological processes.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Ácido Hipocloroso/análise , Ácido Hipocloroso/metabolismo , Substâncias Luminescentes/química , Animais , Linhagem Celular Tumoral , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/toxicidade , Humanos , Inflamação/induzido quimicamente , Inflamação/diagnóstico por imagem , Luciferases/química , Substâncias Luminescentes/síntese química , Substâncias Luminescentes/toxicidade , Masculino , Camundongos , ZimosanRESUMO
Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes/métodos , Animais , Benzotiazóis/metabolismo , Linhagem Celular , Besouros/enzimologia , Células Dendríticas/imunologia , Epitopos , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Linfonodos/citologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fatores de Transcrição NFATC/metabolismo , Ovalbumina/imunologia , Baço/citologia , VacinaçãoRESUMO
The prodrug or caged-luciferin strategy affords an excellent platform for persistent bioluminescence imaging. In the current work, we designed and synthesized ten novel pro-substrates for Renilla luciferase by introducing ester protecting groups of different sizes into the carbonyl group of the free luciferin 1. Taking advantage of intracellular esterases, lipases, and nucleophilic substances, the ester protecting groups were hydrolyzed, resulting in the release of a free luciferin and a bioluminescence signal turn-on. Among the tested pro-substrates, the butyryloxymethyl luciferin 7 exhibited low cytotoxicity and a prolonged luminescence signal both in cellulo and in vivo. Therefore, the butyryloxymethyl luciferin 7 can act as a promising substrate for noninvasive extended imaging in diagnostic and therapeutic fields.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/análise , Medições Luminescentes , Renilla/enzimologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/farmacologia , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Especificidade por SubstratoRESUMO
Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.
Assuntos
Infecções por Acinetobacter/metabolismo , Anemia Ferropriva/metabolismo , Luciferina de Vaga-Lumes/análise , Corantes Fluorescentes/análise , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , 2,2'-Dipiridil/farmacologia , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/patologia , Acinetobacter baumannii/patogenicidade , Acinetobacter baumannii/fisiologia , Anemia Ferropriva/genética , Anemia Ferropriva/patologia , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cátions Bivalentes , Modelos Animais de Doenças , Compostos Férricos/farmacologia , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/síntese química , Corantes Fluorescentes/síntese química , Regulação da Expressão Gênica , Hepcidinas/genética , Hepcidinas/metabolismo , Homeostase/genética , Sobrecarga de Ferro/genética , Sobrecarga de Ferro/patologia , Proteína 1 Reguladora do Ferro/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/metabolismo , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Compostos de Amônio Quaternário/farmacologia , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transdução de Sinais , Transferrina/genética , Transferrina/metabolismoRESUMO
Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues. An ACSL from the nonluminescent beetle Agrypnus binodulus (AbLL) was found to be a second latent luciferase with distinct substrate specificity. Several rigid luciferins emit light with both ACSLs, but styryl luciferin analogues are light-emitting substrates only for AbLL. On the other hand, an ACSL from the luminescent beetle Pyrophorus angustus lacks luciferase activity with all tested analogues, despite its higher homology to beetle luciferases. Further study of ACSLs is expected to shed light on the features necessary for bioluminescence and substrate selectivity.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Luciferases de Vaga-Lume/metabolismo , Animais , Células CHO , Besouros/enzimologia , Cricetulus , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Estrutura Molecular , Especificidade por SubstratoRESUMO
Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/química , Medições Luminescentes , Animais , Células Cultivadas , Vaga-Lumes , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Estrutura Molecular , Engenharia de ProteínasRESUMO
We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.
Assuntos
Luciferina de Vaga-Lumes/metabolismo , Medições Luminescentes , Animais , Linhagem Celular Tumoral , Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/síntese química , Células HEK293 , Halogenação , Humanos , Cinética , Luz , Luciferases de Vaga-Lume/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Five new firefly luciferin (1) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains (2-4) and heterocyclic rings derived from amino acids (5 and 6) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin (6), possessing a pyrroline-substituted benzothiazole structure, had bioluminescence (BL) activity (λmax =547â nm). Results of bioluminescence studies with AMP-carboluciferin (AMP=adenosine monophosphate) and AMP-firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin-luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Substâncias Luminescentes/química , Monofosfato de Adenosina/química , Animais , Benzotiazóis/química , Luciferina de Vaga-Lumes/síntese química , Luciferina de Vaga-Lumes/metabolismo , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/administração & dosagem , Substâncias Luminescentes/síntese química , Medições Luminescentes , Camundongos , Camundongos Transgênicos , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2-a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence ("glow" systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2-a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.
Assuntos
Luciferina de Vaga-Lumes/síntese química , Imidazóis/química , Luciferases/metabolismo , Pirazinas/química , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/química , Humanos , Imidazóis/síntese química , Luminescência , Medições Luminescentes , Proteínas Luminescentes/química , Pirazinas/síntese químicaRESUMO
An amino acid ester derivative of luciferin (valoluc) was synthesized to mimic the transport and activation of valacyclovir. This molecule was characterized in vitro for specificity and enzymatic constants, and then assayed in two different, physiologically-relevant conditions. It was demonstrated that valoluc activation is sensitive to the same cellular factors as valacyclovir and thus has the potential to elucidate the dynamics of amino acid ester prodrug therapies in a functional, high-throughput manner.
Assuntos
Hidrolases de Éster Carboxílico/análise , Luciferina de Vaga-Lumes/análogos & derivados , Luciferina de Vaga-Lumes/química , Proteínas de Membrana Transportadoras/análise , Bactérias/metabolismo , Transporte Biológico , Hidrolases de Éster Carboxílico/metabolismo , Luciferina de Vaga-Lumes/síntese química , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Hidrólise , Luminescência , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Molecular , Especificidade da EspécieRESUMO
A selenium analogue of amino-D-luciferin, aminoseleno-D-luciferin, is synthesized and shown to be a competent substrate for the firefly luciferase enzyme. It has a red-shifted bioluminescence emission maximum at 600 nm and is suitable for bioluminescence imaging studies in living subjects.
Assuntos
Luciferina de Vaga-Lumes/química , Selênio/química , Animais , Linhagem Celular Tumoral , Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/síntese química , Humanos , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Cintilografia , Especificidade por Substrato , Transplante HeterólogoRESUMO
Firefly luciferase utilizes the chemical energy of ATP and oxygen to convert its substrate, D-luciferin, into an excited-state oxyluciferin molecule. Relaxation of this molecule to the ground state is responsible for the yellow-green light emission. Synthetic cyclic alkylaminoluciferins that allow robust red-shifted light emission with the modified luciferase Ultra-Glo are described. Overall light emission is higher than that of acyclic alkylaminoluciferins, aminoluciferin, and the native substrate D-luciferin.
Assuntos
Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/química , Luz , Luciferases de Vaga-Lume/metabolismo , Substâncias Luminescentes/química , Trifosfato de Adenosina/química , Animais , Benzotiazóis/química , Luciferina de Vaga-Lumes/síntese química , Luciferases de Vaga-Lume/química , Substâncias Luminescentes/síntese química , Estrutura Molecular , Oxigênio/química , Estereoisomerismo , Especificidade por SubstratoRESUMO
A set of stable and efficient photoactivable bioluminescent probes for imaging luciferase activity has been developed, which displayed robust bioluminescent signals upon brief UV illumination in buffer, cells and living animals.