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Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue M2 was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λmax values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with M2-specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.
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Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-ß (Aß), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aß amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.
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Oxígeno , Humanos , Oxígeno/química , Oxígeno/metabolismo , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/química , Amiloide/metabolismo , Amiloide/química , Estructura Molecular , AnimalesRESUMEN
Single-chain multicolor-reporter imaging templates were developed for the subcellular localization of molecular events in mammalian cells. The templates were constructed by tandem linkage of fluorescent protein variants - fused with luciferases and the subcellular localization signal peptides. The templates simultaneously reported steroid hormonal activities at different optical spectra in the subcellular compartments. The templates contribute to the expansion of a toolbox of optical probes for subcellular localization of molecular events in intact cells.
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To develop novel drugs for treating T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) which are highly malignant hematological tumors, a series of analogs having a polyenylpyrrole structure of natural compounds (rumbrin and auxarconjugatin B) were synthesized and investigated their structure-activity relationships (SAR) of in vitro anti-T-ALL and anti-AML activities. We obtained three findings: (1) introduction of a methyl group at the conjugated polyene terminus enhanced anti-T-ALL activity, (2) analogs with a 3-chloropyrrole moiety had even higher selectivity for T-ALL cells, and (3) some analogs were effective against AML-derived cells. Among the studied compounds, 3-chloro-2-(8-ethoxycarbonylnona-1,3,5,7-tetraenyl) pyrrole 4e was the most promising candidate of T-ALL- and AML-treating drug. This study provides useful structural information for designing novel drugs treating T-ALL and AML.
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T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.
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Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.
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Células Endoteliales , Neoplasias , Ratones , Animales , Células del Estroma , Hipoxia , Hipoxia de la Célula , Inflamación , Imagen Óptica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Albumin assays in serum are important for the prognostic assessment of many life-threatening diseases, such as heart failure, liver disease, malnutrition, inflammatory bowel disease, infections, and kidney disease. In this study, synthetic coelenterazine (CTZ) indicators are developed to quantitatively illuminate human and bovine serum albumins (HSA and BSA) with high specificity. Their functional groups were chemically modified to specifically emit luminescence with HSA and BSA. The CTZ indicators were characterized by assaying the most abundant serum proteins and found that the CTZ indicators S6 and S6h were highly specific to HSA and BSA, respectively. Their colors were dramatically converted from blue, peaked at 480 nm, to yellowish green, peaked at 535 nm, according to the HSA-BSA mixing ratios, wherein the origins and mixing levels of the albumins can be easily determined by their colors and peak positions. The kinetic properties of HSA and BSA were investigated in detail, confirming that the serum albumins catalyze the CTZ indicators, which act as pseudo-luciferases. The catalytic reactions were efficiently inhibited by specific inhibitors, blocking the drug-binding sites I and II of HSA and BSA. Finally, the utility of the CTZ indicators was demonstrated through a quantitative imaging of the real fetal bovine serum (FBS). This study is the first example to show that the CTZ indicators specify HSA and BSA with different colors. This study contributes to the expansion of the toolbox of optical indicators, which efficiently assays serum proteins in physiological samples. Considering that these CTZ indicators immediately report quantitative optical signals with high specificity, they provide solutions to conventional technical hurdles on point-of-care assays of serum albumins.
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Albúmina Sérica Bovina , Albúmina Sérica , Humanos , Albúmina Sérica Bovina/química , Albúmina Sérica/química , Imidazoles , Pirazinas , Albúmina Sérica Humana , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors' previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.
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Bioensayo , Sondas Moleculares , Animales , Chlorocebus aethiops , Filogenia , Células COS , Luciferasas/química , Mediciones Luminiscentes/métodos , Mamíferos/metabolismoRESUMEN
Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.
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Sondas Moleculares , Proteínas de Unión a Tacrolimus , Unión Proteica , Luciferasas/genética , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Sirolimus/química , Sirolimus/metabolismoRESUMEN
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.
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Diagnóstico por Imagen , Mediciones Luminiscentes , Animales , Luciferasas/química , Carbocianinas , Transferencia de Energía , Mediciones Luminiscentes/métodos , Mamíferos/metabolismoRESUMEN
A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named "S-series". For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes.
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Imidazoles , Pirazinas , Animales , Ligandos , Luciferasas/química , Imidazoles/química , Pirazinas/química , Mediciones Luminiscentes/métodos , MamíferosRESUMEN
The present study introduces a unique BL signature imaging system with novel CTZ analogues named "C-series." Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.
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Mediciones Luminiscentes , Pirazinas , Mediciones Luminiscentes/métodos , Luciferasas/genética , Indicadores y ReactivosRESUMEN
Bioluminescence (BL) is unique cold body radiation of light, generated by luciferin-luciferase reactions and commonly used in various bioassays and molecular imaging. However, most of the peak emissions of BL populate the blue-yellow region and have broad spectral bandwidths and thus superimpose each other, causing optical cross-leakages in multiplex assays. This study synthesized a new series of coelenterazine (CTZ) analogues, named K-series, that selectively illuminates marine luciferases with unique, blue-shifted spectral properties. The optical property and specificity of the K-series CTZ analogues were characterized by marine luciferases, with K2 and K5 found to specifically luminesce with ALuc- and RLuc-series marine luciferases, respectively. The results confirmed that the luciferase specificity and color variation of the CTZ analogues minimize the cross-leakages of BL signals and enable high-throughput screening of specific ligands in the mixture. The specificity and color variation of the substrates were further tailored to marine luciferases (or single-chain bioluminescent probes) to create a multiplex quadruple assay system with four integrated, single-chain bioluminescent probes, with each probe designed to selectively luminesce only with its specific ligand (first authentication) and a specific CTZ analogue (second authentication). This unique multiplex quadruple bioluminescent assay system is an efficient optical platform for specific and high-throughput imaging of multiple optical markers in bioassays without optical cross-leakages.
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Mediciones Luminiscentes , Imagen Molecular , Ligandos , Luciferasas/química , Mediciones Luminiscentes/métodos , BioensayoRESUMEN
Brain-derived neurotrophic factor (BDNF) plays a crucial role in numerous brain functions, including memory consolidation. Previously, we generated a Bdnf-Luciferase transgenic (Bdnf-Luc) mouse strain to visualize changes in Bdnf expression using in vivo bioluminescence imaging. We successfully visualized activity-dependent Bdnf induction in living mouse brains using a d-luciferin analog, TokeOni, which distributes to the brain and produces near-infrared bioluminescence. In this study, we compared the patterns of bioluminescence signals within the whole body of the Bdnf-Luc mice produced by d-luciferin, TokeOni and seMpai, another d-luciferin analog that produces a near-infrared light. As recently reported, hepatic background signals were observed in wild-type mice when using TokeOni. Bioluminescence signals were strongly observed from the region containing the liver when using d-luciferin and TokeOni. Additionally, we detected signals from the brain when using TokeOni. Compared with d-luciferin and TokeOni, signals were widely detected in the whole body of Bdnf-Luc mice by seMpai. The signals produced by seMpai were strong in the regions containing skeletal muscles in particular. Taken together, the patterns of bioluminescence signals in Bdnf-Luc mice vary when using different luciferase substrates. Therefore, the expression of Bdnf in tissues and organs of interest could be visualized by selecting an appropriate substrate.
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Factor Neurotrófico Derivado del Encéfalo , Luciferinas , Animales , Ratones , Factor Neurotrófico Derivado del Encéfalo/genética , Luciferasas/genética , Luciferasas/metabolismoRESUMEN
The present protocol introduces a visible light bioluminescence imaging (BLI) platform together with 12 novel coelenterazine (CTZ) analogues and luciferase sets. We exemplify to create diverse hues of bioluminescence (BL) ranging from blue to far red with the combination of marine luciferases and the three groups of CTZ analogues. We also show how to characterize the new CTZ analogues in detail such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to artificial luciferases and are completely dark with Renilla luciferase derivatives in contrast. The 3d is highly specific to only NanoLuc. This BL imaging system covering the visible region provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.
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Luz , Mediciones Luminiscentes , Animales , Luciferasas/genética , Luciferasas de Renilla/genética , Mediciones Luminiscentes/métodos , MamíferosRESUMEN
AkaLumine hydrochloride, named TokeOni, is one of the firefly luciferin analogs, and its reaction with firefly luciferase produces near-infrared (NIR) bioluminescence. Prior to studying the bioluminescence mechanism, basic knowledge about the chemical structures, electronic states, and absorption properties of TokeOni at various pH values of solution has to be acquired. In this paper, the absorption spectra for TokeOni and AkaLumine at pH 2-10 were measured. Density functional theory (DFT) calculations, time-dependent DFT calculations, and the vibrational analyses were carried out. The absorption spectra indicate that the chemical forms of TokeOni in solutions are same as those of AkaLumine. The peaks at pH 7-10 in the absorption spectra correspond to the excitation from the ground state of a carboxylate anion of AkaLumine, the peak at pH 2 corresponds to the excitation from the ground state of a carboxylate anion with an N-protonated thiazoline ring and N-protonated dimethylamino group of AkaLumine, and the peak at pH 4 corresponds to the excitation from the ground state of a carboxylate anion with an N-protonated thiazoline ring of AkaLumine.
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Luciérnagas , Luciferina de Luciérnaga , Animales , Aniones , Luciferina de Luciérnaga/química , Concentración de Iones de Hidrógeno , Luciferasas de Luciérnaga/químicaRESUMEN
Bioluminescence reactions are widely applied in optical in vivo imaging in the life science and medical fields. Such reactions produce light upon the oxidation of a luciferin (substrate) catalyzed by a luciferase (enzyme), and this bioluminescence enables the quantification of tumor cells and gene expression in animal models. Many researchers have developed single-color or multicolor bioluminescence systems based on artificial luciferin analogues and/or luciferase mutants, for application in vivo bioluminescence imaging (BLI). In the current review, we focus on the characteristics of firefly BLI technology and discuss the development of luciferin analogues for high-resolution in vivo BLI. In addition, we discuss the novel luciferin analogues TokeOni and seMpai, which show potential as high-sensitivity in vivo BLI reagents.
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Diagnóstico por Imagen , Luciferina de Luciérnaga/química , Luciferasas de Luciérnaga/metabolismo , Mediciones Luminiscentes , AnimalesRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a hardly curable disease with a high relapse rate. 20 analogs were synthesized based on the structures of two kinds of fungi-derived polyenylpyrrole products (rumbrin (1) and auxarconjugatin-B (2)) to suppress the growth of T-ALL-derived cell line CCRF-CEM and tested for growth-inhibiting activity. The octatetraenylpyrrole analog gave an IC50 of 0.27 µM in CCRF-CEM cells, while it did not affect Burkitt lymphoma-derived cell line Raji and the cervical cancer cell line HeLa, or the oral cancer cell line HSC-3 (IC50 > 10 µM). This compound will be a promising compound for developing T-ALL-specific drugs.
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Antineoplásicos/farmacología , Polienos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Pirroles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Polienos/síntesis química , Polienos/química , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
The present study describes a color-tunable imaging portfolio together with twelve novel coelenterazine (CTZ) analogues. The three groups of CTZ analogues create diverse hues of bioluminescence (BL) ranging from blue to far red with marine luciferases. We found that the hue completes the whole color palette in the visible region and shows red-shifted BL with a marine luciferase: for example, Renilla luciferase 8 (RLuc8) and Artificial Luciferase 16 (ALuc16) show 187 nm- and 105 nm-redshifted spectra, respectively, by simply replacing the substrate CTZ with 1d. The optical properties of the new CTZ analogues were investigated such as the kinetic parameters, dose dependency, and luciferase specificity. The 2-series CTZ analogues interestingly have specificity to ALucs and are completely dark with RLuc derivatives, and 3d is highly specific to only NanoLuc. We further determined the theoretical background of the red-shifted BL maximum wavelengths (λBL) values according to the extended π conjugation of the CTZ backbone using Density Functional Theory (DFT) calculations. This color-tunable BL imaging system provides a useful multicolor imaging portfolio that efficiently images molecular events in mammalian cells.
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Biotecnología/métodos , Mediciones Luminiscentes/métodos , Luciferasas de Renilla , Biología Molecular , Óptica y Fotónica/métodosRESUMEN
Bioluminescence imaging (BLI) is useful to monitor cell movement and gene expression in live animals. However, D-luciferin has a short wavelength (560 nm) which is absorbed by tissues and the use of near-infrared (NIR) luciferin analogues enable high sensitivity in vivo BLI. The AkaLumine-AkaLuc BLI system (Aka-BLI) can detect resolution at the single-cell level; however, it has a clear hepatic background signal. Here, to enable the highly sensitive detection of bioluminescence from the surrounding liver tissues, we focused on seMpai (C15H16N3O2S) which has been synthesized as a luciferin analogue and has high luminescent abilities as same as AkaLumine. We demonstrated that seMpai BLI could detect micro-signals near the liver without any background signal. The solution of seMpai was neutral; therefore, seMpai imaging did not cause any adverse effect in mice. seMpai enabled a highly sensitive in vivo BLI as compared to previous techniques. Our findings suggest that the development of a novel mutated luciferase against seMpai may enable a highly sensitive BLI at the single-cell level without any background signal. Novel seMpai BLI system can be used for in vivo imaging in the fields of life sciences and medicine.