RESUMO
G-quadruplex (G4), an unconventional nucleic acid structure, shows polymorphism in its topological morphology. The parallel G4 topology is the most prevalent form in organisms and plays a regulatory role in many biological processes. Designing fluorescent probes with high specificity for parallel G4s is important but challenging. Herein, a supramolecular assembly of the anionic cyanine dye SCY-5 is reported, which selectively identifies parallel G4 topology. SCY-5 can clearly distinguish parallel G4s from other G4s and non-G4s, even including hybrid-type G4s with parallel characteristics. The high specificity mechanism of SCY-5 involves a delicate balance between electrostatic repulsion and π-π interaction between SCY-5 and G4s. Using SCY-5, cellular RNA extracted from peripheral venous blood was quantitatively detected, and a remarkable increase in RNA G4 content in cancer patients compared to healthy volunteers was confirmed for the first time. This study provides new insights for designing specific probes for parallel G4 topology and opens a new path for clinical cancer diagnosis using RNA G4 as a biomarker.
Assuntos
Carbocianinas , Corantes Fluorescentes , Quadruplex G , Neoplasias , Humanos , Carbocianinas/química , Corantes Fluorescentes/química , Neoplasias/diagnóstico , RNA/química , RNA/análiseRESUMO
Noncanonical nucleic acid structures, such as G-quadruplex (G4) and i-Motif (iM), have attracted increasing research interests because of their unique structural and binding properties, as well as their important biological activities. To date, thousands of small molecules that bind to varying G4/iM structures have been designed, synthesized and tested for diverse chemical and biological uses. Because of the huge potential and increasing research interests on G4-targeting ligands, we launched the first G4 ligand database G4LDB in 2013. Here, we report a new version, termed G4LDB 2.2 (http://www.g4ldb.com), with upgrades in both content and function. Currently, G4LDB2.2 contains >3200 G4/iM ligands, â¼28 500 activity entries and 79 G4-ligand docking models. In addition to G4 ligand library, we have also added a brand new iM ligand library to G4LDB 2.2, providing a comprehensive view of quadruplex nucleic acids. To further enhance user experience, we have also redesigned the user interface and optimized the database structure and retrieval mechanism. With these improvements, we anticipate that G4LDB 2.2 will serve as a comprehensive resource and useful research toolkit for researchers across wide scientific communities and accelerate discovering and validating better binders and drug candidates.
Assuntos
Bases de Dados Genéticas , Quadruplex G , Relação Estrutura-Atividade , Sítios de Ligação/genética , Humanos , Ligantes , Simulação de Acoplamento MolecularRESUMO
SO2, a gas signaling molecule, can be produced endogenously in mitochondria. Its hydrolysate, HSO3-, plays a key role in food preservation, cardiovascular relaxation, and other fields, suggesting that it is important to achieve its detection. Here, based on the Michael addition mechanism, four hemicyanine dye fluorescent probes (ETN, ETB, STB, and EIB) were designed and synthesized for responding to HSO3-. We evaluated the reaction ability of different probes with HSO3- and tried to explain the reasons for the significantly different response effects between probes and HSO3- according to the structure-activity relationship. The influence of different substituents of probes on the properties of mitochondria-targeting was also discussed. Finally, we screened out ETN as the optimal HSO3- probe due to its high sensitivity, rapid reactivity, and good mitochondria-targeting, and it could sensitively respond to HSO3- in living cells. The LODs of ETN for HSO3- were calculated by both absorption and fluorescence methods, respectively, which were 2.727 and 0.823 µM. Our work provided valuable references for designing strategies and potential tools for response to SO2 derivatives in biosystems.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Humanos , Carbocianinas , Limite de Detecção , Sulfitos , Células HeLaRESUMO
The material transport and physiological events of mitochondria need to be supported by a suitable microenvironment. For example, high viscosity will seriously hinder material exchange, and SO2, as the precursor of HSO3-, is an endogenous signal molecule that plays a key role in information transmission. It is very important to detect viscosity and HSO3- in mitochondria. Here, we developed a dual-responsive fluorescent probe (named Hcy-NT) to image the changes in mitochondrial viscosity and HSO3- in a "killing two birds with one stone" manner. Hcy-NT showed an OFF-ON fluorescence signal for the increase in cell viscosity induced by nystatin, while an ON-OFF fluorescence signal for intracellular and endogenous HSO3-. Its limits of detection for HSO3- were calculated by both absorption and fluorescence methods, which were 1.200 and 1.291 µM, respectively. This work provides a valuable tool for the study of viscosity and HSO3- related physiological processes and the diagnosis of potential diseases.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Humanos , Corantes Fluorescentes/toxicidade , Viscosidade , Fluorescência , Células HeLaRESUMO
Antibiotic abuse is considered a serious problem affecting human health, necessitating that great attention be paid to explore robust, simple and sensitive methods for rapid evaluation. In this paper, we developed a fluorescent aptasensor for visual and real-time kanamycin detection by taking advantage of the label-free strategy based on H-aggregate disassembly of a chiral cyanine dye induced by a G-quadruplex aptamer. The good sensitivity and selectivity enabled this aptasensor to have a detection limit as low as 43 nM and have high specificity for kanamycin recognition. Furthermore, this assay was successfully applied for the detection of kanamycin in lake water and urine with excellent recoveries.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Humanos , Canamicina , Lagos , Corantes Fluorescentes , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
Glutaminase 1 (GLS1) has recently been reported to be expressed in microglia and plays a crucial role in neuroinflamation. Significantly increased level of GLS1 mRNA expression together with neuroinflammation pathway were observed in postmortem prefrontal cortex from depressed patients. To find out the function of microglial GLS1 in depression and neuroinflammation, we generated transgenic mice (GLS1 cKO), postnatally losing GLS1 in microglia, to detect changes in the lipopolysaccharide (LPS)-induced depression model. LPS-induced anxiety/depression-like behavior was attenuated in GLS1 cKO mice, paralleled by a significant reduction in pro-inflammatory cytokines and an abnormal microglia morphological phenotype in the prefrontal cortex. Reduced neuroinflammation by GLS1 deficient microglia was a result of less reactive astrocytes, as GLS1 deficiency enhanced miR-666-3p and miR-7115-3p levels in extracellular vesicles released from microglia, thus suppressing astrocyte activation via inhibiting Serpina3n expression. Together, our data reveal a novel mechanism of GLS1 in neuroinflammation and targeting GLS1 in microglia may be a novel strategy to alleviate neuroinflammation-related depression and other disease.
Assuntos
Glutaminase , Microglia , Animais , Depressão , Glutaminase/genética , Glutaminase/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Doenças NeuroinflamatóriasRESUMO
Owing to its potential biological relevance, DNA G-quadruplex has been considered as a prospective anti-cancer target. Some known G-quadruplex-interactive N-containing compounds with low cytotoxicity have become prospective anticancer drugs. Here we reported a new type of N-containing alkaloids 3,8a-disubstituted indolizinones, and investigated their substituent effects at 3- and 8a-positions in targeting to DNA c-myc G-quadruplex. And then we used 3-naphtyl-8a-(pyridin-2-yl) substrate I8 as an example, and investigated its ability in targeting to DNA parallel G-quadruplexes in vitro.
Assuntos
Antineoplásicos/química , DNA de Neoplasias/análise , Indolizinas/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Humanos , Indolizinas/síntese química , Indolizinas/farmacologia , Estrutura Molecular , Proteínas Proto-Oncogênicas c-myc/análise , Proteínas Proto-Oncogênicas c-myc/genética , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in living cells can provide valuable insights into the relationship between G-quadruplex formation and its cellular consequences. However, the probes capable of detecting DNA G-quadruplexes in living cells are still very limited. Herein, we reported a new fluorescent probe, IMT, for real-time visualization of DNA G-quadruplex structures in living cells. Using IMT as a fluorescent indicator, the quantity changes of DNA G-quadruplex at different points in time during continuous cellular progression responding to Aphidicolin and Hydroxyurea treatment have been directly visualized. Our data demonstrate that IMT will be a valuable tool for exploring DNA G-quadruplexes in live cells. Further application of IMT in fluorescence imaging may reveal more information on the roles of DNA G-quadruplexes in biological systems.
Assuntos
DNA/química , Corantes Fluorescentes/química , Quadruplex G/efeitos dos fármacos , Afidicolina/química , Linhagem Celular Tumoral , Células HeLa , Humanos , Hidroxiureia/química , Microscopia de Fluorescência , Espectrometria de FluorescênciaRESUMO
Molecular computation is increasingly attractive as a tool for medical and biological research because of its programmability and controllability. Herein, a novel visibly observable supramolecular system that can execute multi-level logic functions on a uniform platform was constructed. By employing some programming factors, we succeeded in not only constructing a whole set of contrary logic pairs, but also building up a logic network that can implement advanced functions. Further, the platform is applied to sense thiols in specific environments. The developed method can efficiently filter signals of thiols in intracellular conditions and measure cysteine levels quantitatively in serum conditions. The visual readout makes the method particularly suitable for point-of-care testing. The supramolecule-based platform illustrates not only an incremental advance for the construction of programmable molecular logic systems, but also viable applications in intelligent thiol analysis.
RESUMO
The design of DNA-based logic circuits has become an active research field in DNA nanotechnology and holds great potential in intelligent bioanalysis. To date, although many DNA-based logic systems have been realized, the implementation of advanced logic functions is still challenging, especially with simple and homogeneous compositions. Herein, by integrating two DNA tetraplex structures (G-quadruplex and i-motif), a completely label-free logic platform with high scalability was established, with which a series of advanced functions were realized, including arithmetic (adders and subtractors) and nonarithmetic ones (majority and dual-transfer gates). Furthermore, the platform was also applied as an intelligent biosensor to coanalyze two cancer-related micro-RNAs with high sensitivities and specificities. Considering the excellent versatility, expandability, and biocompatibility, the platform may promote the development of DNA computing and hold great potential in multiparameter sensing and medical diagnosis.
Assuntos
Técnicas Biossensoriais/métodos , Computadores Moleculares , Quadruplex G , MicroRNAs/análise , Nanoestruturas/química , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Fluorescência , Humanos , Nanotecnologia , Espectrometria de Fluorescência/métodosRESUMO
The lead (Pb) hazard is not only in connection with the concentration of Pb2+ but also closely related to the ambience which affects its mobility and the synergistic toxicity with other ions. However, most of the existent methods focus highly on detecting Pb2+ concentration accurately but can seldom reflect the pollution-related information in actual samples, thereby limiting their pragmatic applications. In this work, a DNA-supramolecule logic platform was established, which can be configurated to implement three information process functions and act as three unique intelligent sensors of Pb. The demultiplexer that can split signal flow was used to determine Pb2+ in different pH conditions; the multiplexer that can alternate signal channels was applied to detect Pb2+ or Ag+ selectively; and the decoder that can extract information was utilized to test Pb2+ and the coexisted Ni2+ simultaneously. All three intelligent sensors based on the logic prototypes present practicable sensitivities and specificities. Considering its flexibility, scalability, and reconfigurability, we believe the logic platform may provide new solutions to process sophisticated information and implement intelligent analysis in environmental monitoring, biochemical detecting, and medical diagnosis.
Assuntos
Técnicas Biossensoriais , Técnicas de Química Analítica/métodos , DNA/análise , Chumbo/química , Nanopartículas Metálicas/química , Prata/análiseRESUMO
Plenty of molecular circuits with specific functions have been developed; however, logic units with reconfigurability, which could simplify the circuits and speed up the information process, are rarely reported. In this work, we designed a novel reconfigurable logic unit based on a DNA-templated, potassium-concentration-dependent, supramolecular assembly, which could respond to the input stimuli of H+ and K+ . By inputting different concentrations of K+ , the logic unit could implement three significant functions, including a half adder, a half subtractor, and a 2-to-4 decoder. Considering its reconfigurable ability and good performance, the novel prototypes developed here may serve as a promising proof of principle in molecular computers.
Assuntos
Computadores Moleculares , DNA/química , Lógica , Potássio/análise , Replicação do DNA , Estrutura MolecularRESUMO
As key molecules in most biological pathways, proteins physically contact one or more biomolecules in a highly specific manner. Several driving forces (i.e., electrostatic and hydrophobic) facilitate such interactions and a variety of methods have been developed to monitor these processes both in vivo and in vitro. In this work, a new method is reported for the detection of protein interactions by visualizing a color change of a cyanine compound, a supramolecule complex of 3,3-di-(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt (MTC). Nuclear magnetic resonance (NMR) studies suggest that the hydrophobic nature of the protein surfaces drives MTC into different types of aggregates with distinct colors. When proteins interact with other biomolecules, the hydrophobic surface of the complex differs, resulting in a shift in the form of MTC aggregation, which results in a color change. As a result, this in vitro method has the potential to become a rapid tool for the confirmation of protein-biomolecule interactions, without the requirements for sophisticated instrumentation or approaches.
Assuntos
Carbocianinas/química , Colorimetria , Proteínas/química , Carbocianinas/metabolismo , DNA/química , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
BACKGROUND: Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. METHODS: Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). RESULTS: Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. CONCLUSIONS: The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. GENERAL SIGNIFICANCE: The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.
Assuntos
Núcleo Celular/metabolismo , Corantes Fluorescentes/química , Quadruplex G , Sondas Moleculares/química , Tiazóis/química , Benzotiazóis , Núcleo Celular/química , Dicroísmo Circular , Humanos , Células MCF-7 , Microscopia de Fluorescência , Espectrometria de FluorescênciaRESUMO
Thrombin plays a critical role in hemostasis and hemolysis. It is of high importance to develop a system toward thrombin detection with high sensitivity and high selectivity for both research and clinical diagnosis applications. In this paper, we developed a thrombin detection assay by taking advantage of the novel signal amplified strategy based on the chiral supramolecular assembly in physiological K+ background. This assay could detect thrombin as low concentration as about 2 pM and provided a highly specific selectivity among several common interferences. Furthermore, the assay can discriminate thrombin from other nonspecific analogous proteins with high selectivity and can be used to detect thrombin in diluted real human serum samples, which suggested its great potential for rapid detection of thrombin in the clinic.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Limite de Detecção , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Carbocianinas/química , Humanos , Estereoisomerismo , Trombina/química , Trombina/metabolismoRESUMO
A fluorescent cationic benzothiazole dye that selectively targets a G-quadruplex aptamer was designed and synthesized as a K+ sensor. The K+-driven aptamer sensor is based on the strategy of conformational transition from single-stranded DNA to G-quadruplex structure, leading to an amplified fluorescence signal in the reporter. This fluorescent sensor displayed high selectivity for K+, suggesting great potential for practical applications.
RESUMO
RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.
Assuntos
Benzotiazóis , Carbocianinas , Corantes Fluorescentes , Quadruplex G , RNA/química , Benzotiazóis/química , Carbocianinas/química , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Humanos , Microscopia Confocal , QuinolinasRESUMO
The G-quadruplexes located in the P1 promoter of B-cell lymphoma-2 (Bcl-2) gene are implicated to regulate Bcl-2 expression. Here, we designed a new pyridostatin analog named PDF, which exhibited high specificity and stabilizing effect toward G-quadruplexes. The luciferase assay demonstrated that PDF could significantly suppress Bcl-2 transcriptional activation in human laryngeal squamous carcinoma cells (Hep-2) cells. Besides, PDF also induced cell apoptosis in vitro assays. These results provide an excellent G-quadruplex specific ligand as an efficient Bcl-2 inhibitor. These results also implicate that PDF may be a potential anticancer drug to head neck cancer.
Assuntos
Aminoquinolinas/química , Aminoquinolinas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Quadruplex G/efeitos dos fármacos , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , DNA/genética , Humanos , Neoplasias Laríngeas/tratamento farmacológico , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/patologia , Laringe/efeitos dos fármacos , Laringe/metabolismo , Laringe/patologia , Ativação Transcricional/efeitos dos fármacosRESUMO
It is found that G-quadruplexes have important functions in biological systems, such as gene expression. Molecules which can stabilize the G-quadruplex structure may have potential application in regulating the expression of gene. A series of methylazacalix[n]pyridine (n=4, 6, 7, 8, 9) has been tested to stabilize the intermolecular human telomeric G-quadruplex (T12 and H12), intramolecular TBA, c-kit and bcl-2 G-quadruplex by CD denaturation experiments. The results showed that only methylazacalix[6]pyridine (MACP6) can stabilize the intermolecular G-quadruplex formed from the 12bp human telomere. Further studies evidenced that the shape-complementary binding mode was what contributed to the interaction between MACP6 and T12 G-quadruplex.
Assuntos
Calixarenos/farmacologia , DNA/química , Excipientes/farmacologia , Quadruplex G/efeitos dos fármacos , Telômero/efeitos dos fármacos , Dicroísmo Circular , Humanos , Simulação de Acoplamento Molecular , Desnaturação de Ácido Nucleico/efeitos dos fármacos , Telômero/químicaRESUMO
A novel fluorescent dye, 1-(2-hydroxyethyl)-4-((E)-2-(3-benzothiazol-2yl-9-ethyl-carbazole-3yl)vinyl) pyridinium bromide, was synthesized for determination of protein and its structure was characterized by (1)H NMR. Photophysics of the new probe in different solvents has been delineated in this paper, the new fluorescent molecular dye exhibited a large stokes-shifted and fluorescence quantum yields in organic solvent. The photostability and thermostability of the new dye were also studied and the results suggested the stable was excellent. The interactions of the dye with bovine serum albumin (BSA) , Human serumal bumin (HSA) and calf thymus deoxyribonucleic acid (ctDNA) were studied by fluorescence and absorption spectroscopy. The binding constant for BSA, HSA and DNA were calculated to be 8.91 × 10(7), 1.86 × 10(6) and 2.9 × 10(4), respectively. The experimental results indicated a potential value of the new dye for biomarker.