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1.
Chem Commun (Camb) ; 55(67): 9967-9970, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367705

RESUMO

Herein, a seesaw ratiometric (SR) probe is designed which integrates fluorescence and surface enhanced Raman scattering (SERS) technology. Fluorescence imaging enables tracking of the spatiotemporal dynamic behaviour of telomerase. Meanwhile, SERS reverse ratiometric measurement can enable sensitive detection of telomerase activity in single living cells.


Assuntos
Corantes/química , Imagem Óptica/métodos , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Telomerase/metabolismo , DNA/química , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Humanos , Células MCF-7 , Nanopartículas Metálicas/química
2.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
3.
Analyst ; 144(13): 3999-4005, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31172144

RESUMO

Micrococcal nuclease (MNase) is an extracellular endonuclease of Staphylococcus aureus (S. aureus). It digests single stranded nucleic acid. The presence of MNase is the gold standard to identify S. aureus and its content. The present study reports the ultrahigh sensitive and selective fluorescence platform for MNase detection, designed and developed based on the surface energy transfer mechanism. A "proof of concept" is being developed based on monoclonal antibody-conjugated quantum dots (mAb-QDs), wherein mAb-QDs act as donors and graphene oxide (GO) acts as an acceptor. mAb-QDs in close proximity to GO undergo adsorption due to weak affinity between them and this results in fluorescence quenching by the transfer of surface energy from mAb-QDs to GO. During sensing, a much stronger affinity of mAb-QDs towards MNase inhibits the energy transfer to GO and this allows the regaining of fluorescence. Immobilized mAb-QDs on nitrocellulose membrane strips were fabricated and tested for "ON-OFF-ON" sensing of MNase. The limit of detection for fluorescence based assay and strips is found to be 0.3 ng mL-1 and 0.5 ng mL-1, respectively. The developed strips were applied on real samples for the detection of S. aureus.


Assuntos
Corantes Fluorescentes/química , Grafite/química , Nuclease do Micrococo/análise , Pontos Quânticos/química , Staphylococcus aureus/química , Anticorpos Monoclonais/química , Técnicas Biossensoriais/métodos , Colódio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção , Membranas Artificiais , Nanocompostos , Estudo de Prova de Conceito , Sensibilidade e Especificidade , Staphylococcus aureus/enzimologia
4.
Biosens Bioelectron ; 138: 111314, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096114

RESUMO

FÖrster resonance energy transfer (FRET)-based biosensors have achieved great success for biological applications. However, what is not extensively appreciated is the growing role as versatile FRET biosensors within a similar biological context. This review provides a brief introduction of recent advances in principle, the designing strategies and kinds of applications of FRET biosensors. For each FRET biosensor, the appropriate background and fabrication is explained before studying their related applications. The prominent roles of nanomaterials are present in the development of more sensitive and specific FRET biosensors. Finally, the challenges and outlooks of FRET biosensors are emphasized.


Assuntos
Técnicas Biossensoriais/métodos , Animais , Técnicas Biossensoriais/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Nanoestruturas/química
5.
Anal Chim Acta ; 1071: 70-77, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31128757

RESUMO

In this study, a simple paper-based aptasensor has been developed for the ultrasensitive detection of lead (Pb2+) ion within about 10 min. The aptasensor has been successfully designed by taking advantages of the Förster Resonance Energy Transfer (FRET) process and the super fluorescence quenching property of graphene oxide (GO) sheet. The sensing mechanism of the aptasensor is based on the conformational switch of the Pb2+-specific aptamer from a random coil to a G-quadruplex structure. An injection of Pb2+ on the paper-based platform induces the release of the specific aptamer from the GO surface that recovers the fluorescence emission. Under the optimal experimental conditions, there is a good linear relationship between the fluorescence recovery and the Pb2+concentration in the ranges of 5-70 pM and 0.07-20 nM. Moreover, the aptasensing array exhibits a high sensitivity to Pb2+ with an ultra-low detection limit of 0.5 pM. The developed aptasensor has been successfully applied to determine Pb2+ in tap water, lake water, milk, and human blood serum. The paper-based aptasensor can be efficiently utilized to detect other metal ions and biological molecules by substituting target specific aptamer.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Chumbo/sangue , Papel , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Água Potável/análise , Fluorescência , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Contaminação de Alimentos/análise , Quadruplex G/efeitos dos fármacos , Grafite/química , Lagos/análise , Limite de Detecção , Leite/química
6.
Talanta ; 201: 330-334, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122431

RESUMO

A novel ratiometric fluorescence probe for hypochlorous acid was constructed by coumarin and pyridinium fluorophore based on the Forster resonance energy transfer (FRET) and intramolecular charge transfer (ICT) platform. In this ICT/FRET system, the energy transfer efficiency is high to 94.3%. Moreover, the probe could respond to hypochlorous acid with high selectivity and sensitivity, and exhibited a large Stokes shift. It was interesting to find that the probe could recognize hypochlorous acid via a new mechanism, in which the α-position of carbonyl group was oxidized to form a diketone derivative. More importantly, the probe was successfully applied to the ratiometric imaging of both exogenous and endogenous hypochlorous acid in living RAW 264.7 cells, with low toxicity and high photo-stability.


Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Ácido Hipocloroso/metabolismo , Compostos de Piridínio/química , Animais , Cumarínicos/síntese química , Cumarínicos/efeitos da radiação , Desenho de Drogas , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Concentração de Íons de Hidrogênio , Luz , Limite de Detecção , Camundongos , Imagem Óptica/métodos , Compostos de Piridínio/síntese química , Compostos de Piridínio/efeitos da radiação , Células RAW 264.7
7.
Talanta ; 201: 82-89, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122464

RESUMO

Using chloroauric acid as precursor and ß-cyclodextrin (ß-CD) as reducing agent and stabilizer, ß-CD@AuNPs with negative charge were synthesized by one-step colloidal synthesis method. The positive charged carbon quantum dots (CQDs) were synthesized by one-step of sonication of cetylpyridinium chloride. Under the role of static electricity, the fluorescence resonance energy transfer (FRET) occurred between CQDs and ß-CD@AuNPs. CQDs and ß-CD@AuNPs served as the fluorescence energy donors and receptors, respectively, i.e., the fluorescence of CQDs was turned off by ß-CD@AuNPs. Based on the specific host-guest recognition between the inner cavity of ß-CD and cholesterol, CQDs was replaced by cholesterol, the FRET could be interrupted, and then the fluorescence of CQDs was turned on. A good linear relationship between cholesterol concentration (10-210 µmol L-1) and fluorescence intensity was obtained and the LOD was 343.48 nmol L-1. Because of excellent fluorescence quenching ability of FRET, the analytical performance (including LOD and linear scope) of such a turn off-on fluorescent nanosensor (e.g., CQDs/ß-CD@AuNPs) was better than nanosensor only via competitive host-guest recognition (e.g., ß-CD functionalized CQDs). The synergistic effect of competitive host-guest recognition and FRET was proved. Because of selective recognition, ultrasensitive, wide linear range, and strong anti-interference ability, CQDs/ß-CD@AuNPs as a turn off-on fluorescent nanosensor was developed to determine cholesterol in porcine serum.


Assuntos
Colesterol/sangue , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Pontos Quânticos/química , beta-Ciclodextrinas/química , Animais , Carbono/química , Carbono/efeitos da radiação , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Limite de Detecção , Tamanho da Partícula , Pontos Quânticos/efeitos da radiação , Suínos , Raios Ultravioleta
8.
Nat Commun ; 10(1): 2104, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068591

RESUMO

Protein-induced fluorescence enhancement (PIFE) is a popular tool for characterizing protein-DNA interactions. PIFE has been explained by an increase in local viscosity due to the presence of the protein residues. This explanation, however, denies the opposite effect of fluorescence quenching. This work offers a perspective for understanding PIFE mechanism and reports the observation of a phenomenon that we name protein-induced fluorescence quenching (PIFQ), which exhibits an opposite effect to PIFE. A detailed characterization of these two fluorescence modulations reveals that the initial fluorescence state of the labeled mediator (DNA) determines whether this mediator-conjugated dye undergoes PIFE or PIFQ upon protein binding. This key role of the mediator DNA provides a protocol for the experimental design to obtain either PIFQ or PIFE, on-demand. This makes the arbitrary nature of the current experimental design obsolete, allowing for proper integration of both PIFE and PIFQ with existing bulk and single-molecule fluorescence techniques.


Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Endonucleases Flap/química , Endonucleases Flap/isolamento & purificação , Endonucleases Flap/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem , Proteínas Virais/química , Proteínas Virais/isolamento & purificação , Proteínas Virais/metabolismo
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 217: 101-106, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30928834

RESUMO

The development of a simple, rapid and sensitive sensor to detect copper ion is very important for environmental detection. Here, we constructed a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) between copper resistance operon coded protein C (CopC), a copper binding protein, and dansyl chloride (DNS-Cl) to selectively detect copper ion. At alkaline conditions, DNS-Cl was contently attached to CopC forming biosensor of DNS-Cl/CopC, in which fluorescence emission of CopC at 320nm was absorbed by DNS-Cl. After binding with copper ion, the fluorescence of DNS-Cl was quenched significantly to 47%. Within the range of 0.04-11µM (R=0.989), the good linearity was obtained and the detection limit reached to 7nM. More importantly, the biosensor of DNS-Cl/CopC has been successfully used to detecting of copper ion level in purified water, tap water and waste water drained from steel mill. And the results were consistent well with those obtained from the inductively coupled plasma mass spectrometry. Therefore, the established biosensor DNS-Cl/CopC is a creditable method to detect copper ion with high sensitivity and selectivity, which can be utilized as a powerful tool to monitor copper pollution in the environments.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Cobre/análise , Compostos de Dansil/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção
10.
Spectrochim Acta A Mol Biomol Spectrosc ; 217: 247-255, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30947133

RESUMO

The applications of metallic nanoclusters and nanoparticles in biological sensing have attracted special attention owing to their optical interaction based on fluorescence and surface plasmon resonance (SPR). In this work, we designed a fluorescent nanoprobe for the determination of L-cysteine (L-Cys) based on fluorescence resonance energy transfer (FRET) from gold­silver bimetallic nanoclusters (Au-Ag NCs) to gold nanorods (AuNRs). Firstly, the negatively charged Au-Ag NCs protected by bovine serum albumin (BSA) are directly adsorbed on the surface of the positively charged AuNRs through electrostatic interaction, and the FRET effect leads to distinct fluorescence quenching of Au-Ag NCs at 615nm. The SPR wavelength of AuNRs is dependent on the aspect ratio, so the SPR of AuNRs could be tuned to have a better spectral overlap with fluorescence of Au-Ag NCs, which enhances the fluorescence quenching effect. Because the SH group of L-Cys has an affinity with gold, the addition of L-Cys can result in the release of Au-Ag NCs from the surface of AuNRs via forming AuS bonds. Thus, the introduction of L-Cys could effectively restore the fluorescence emission of the AuNRs/Au-Ag NCs system because of the restraint of FRET effect. Under the optimized conditions, the fluorescence recovery of AuNRs/Au-Ag NCs probe exhibits a linear response to L-Cys concentration ranging from 5 to 100µM, and the corresponding theoretical detection limit (LOD) is 1.73µM. Meanwhile, this method displays excellent sensitivity and selectivity for L-Cys over other amino acids, and it has been successfully applied to detect L-Cys in real samples.


Assuntos
Cisteína/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Nanopartículas Metálicas/química , Nanotubos/química , Prata/química , Urina/química , Técnicas Biossensoriais/métodos , Fluorescência , Limite de Detecção
11.
Methods Mol Biol ; 1979: 409-421, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028651

RESUMO

Recent fluorescence microscopy allows for high-throughput acquisition of 5D (X, Y, Z, T, and Color) images in various targets such as cultured cells, 3D spheroid/organoid, and even living tissue with single-cell resolution. The technology is considered promising to augment insights on heterogeneous features of both physiological and pathological cell phenotypes, for instance, distinct responses of cancer cells to anticancer drug treatment. Here we overview microscopic applications to capture live cell events for different types of targets, together with a couple of proof of concepts. The 2D live imaging will be exemplified by a FRET-based time-lapse cultured cell imaging, and 3D tissue imaging protocol will be complemented with a method for mouse skin live imaging.


Assuntos
Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Análise de Célula Única/métodos , Animais , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/análise , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Camundongos , Microscopia de Fluorescência/instrumentação , Imagem Óptica/instrumentação , Análise de Célula Única/instrumentação , Imagem Corporal Total/instrumentação , Imagem Corporal Total/métodos
12.
Methods Mol Biol ; 1973: 251-260, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016707

RESUMO

A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site's U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor's signal with concomitant restoration of the donor's emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.


Assuntos
Aminoglicosídeos/metabolismo , Cumarínicos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , RNA Bacteriano/metabolismo , Uridina/metabolismo , Aminoglicosídeos/química , Cumarínicos/química , Ligantes , RNA Bacteriano/química , Uridina/química
13.
Methods Mol Biol ; 1947: 137-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969414

RESUMO

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Hidrazinas/química , Fragmentos de Peptídeos/química , Receptores Acoplados a Proteínas-G/metabolismo , Acilação , Fluorescência , Humanos , Ligantes , Fragmentos de Peptídeos/metabolismo , Receptores Acoplados a Proteínas-G/química
14.
Methods Mol Biol ; 1947: 151-168, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969415

RESUMO

Although G protein-coupled receptor (GPCR) oligomerization is a matter of debate, it has been shown that the nature of the GPCR partners within the oligomers can influence the pharmacological properties of the receptors. Therefore, finding specific ligands for homo- or hetero-oligomers opens new perspectives for drug discovery. However, no efficient experimental strategy to screen for such ligands existed yet. Indeed, conventional binding strategies do not discriminate ligand binding on GPCR monomers, homo- or hetero-oligomers. To address this issue, we recently developed a new assay based on a time-resolved FRET method that is easy to implement and that can focus on ligand binding specifically on the hetero-oligomer.


Assuntos
Bioensaio/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Transdução de Sinais
15.
Methods Mol Biol ; 1947: 199-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969418

RESUMO

G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imunofluorescência/métodos , Luciferases de Renilla/metabolismo , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Fluorescência , Humanos , Microscopia de Fluorescência
16.
Methods Mol Biol ; 1947: 217-237, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969419

RESUMO

A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlying cAMP change. In this chapter, we provide examples of the problems that may arise when using normalized FRET data and present a method based on the conversion of FRET ratio changes into actual cAMP concentrations that mitigates these issues.


Assuntos
Técnicas Biossensoriais/métodos , AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Humanos , Transdução de Sinais
17.
Analyst ; 144(10): 3216-3220, 2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-30984925

RESUMO

A DSN-RNAse-TdT-T7 exo probing system allows the detection of miRNA 21 with very high sensitivity (LOD = 2.57 fM) and selectivity-the result of (i) avoiding the false-positive signal from miRNA reacting with TdT polymerase and (ii) signal amplification occurring through a FRET-breaking mechanism involving T7 exo.


Assuntos
DNA Nucleotidilexotransferase/química , Exodesoxirribonucleases/química , MicroRNAs/sangue , Ribonucleases/química , Bacteriófago T7/enzimologia , Sondas de DNA/síntese química , Sondas de DNA/genética , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA Mensageiro/genética
18.
Chem Commun (Camb) ; 55(35): 5095-5098, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30957824

RESUMO

Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants.


Assuntos
Corantes Fluorescentes/química , Triptofano/análogos & derivados , Triptofano/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Proteínas de Membrana/síntese química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Triptofano/síntese química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
19.
Chem Commun (Camb) ; 55(35): 5131-5134, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30973555

RESUMO

A novel approach to program target-responsive devices by incorporating the split G4 motifs in a DNA nanocage has been developed. The rigid prism outcompetes the flexible one in reaction kinetics and signal/background ratios, which can be easily internalized by cells and successfully applied in microRNA imaging in live cells.


Assuntos
DNA/química , Quadruplex G , MicroRNAs/análise , Nanopartículas/química , Benzotiazóis/química , Linhagem Celular Tumoral , DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Substâncias Intercalantes/química , Cinética , MicroRNAs/genética , Microscopia Confocal/métodos , Hibridização de Ácido Nucleico
20.
Nat Commun ; 10(1): 1855, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015431

RESUMO

DHX36 is a DEAH-box helicase that resolves parallel G-quadruplex structures formed in DNA and RNA. The recent co-crystal structure of DHX36 bound G4-DNA revealed an intimate contact, but did not address the role of ATP hydrolysis in G4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 displays ATP-independent unfolding of G4-RNA followed by ATP-dependent refolding, generating a highly asymmetric pattern of activity. Interestingly, DHX36 refolds G4-RNA in several steps, reflecting the discrete steps in forming the G4 structure. We show that the ATP-dependent activity of DHX36 arises from the RNA tail rather than the G4. Mutations that perturb G4 contact result in quick dissociation of the protein from RNA upon ATP hydrolysis, while mutations that interfere with binding the RNA tail induce dysregulated activity. We propose that the ATP-dependent activity of DHX36 may be useful for dynamically resolving various G4-RNA structures in cells.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Quadruplex G , Dobramento de RNA , RNA/metabolismo , RNA Helicases DEAD-box/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Microscopia de Fluorescência/métodos , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica/genética , RNA/química , Imagem Individual de Molécula/métodos
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