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1.
Mikrochim Acta ; 188(8): 283, 2021 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-34341883

RESUMEN

Tumor exosomes that inherit specific molecules from their parent cells are emerging as ideal biomarkers in cancer diagnostics. Most currently available exosome isolation and detection methods are time-consuming and non-specific; thus, rapid and specific exosome detection methods are needed both clinically and in research. Here, a dual-functional platform is reported composed of reversible conjunction and "off-on" signal responses. Fe3O4@SiO2@TiO2 particles with high affinity were applied to capture exosomes, and model exosomes could be isolated from solution within 20 min with a capture efficiency of 91.5%. An "on-off" fluorescence response PSMA aptasensor was constructed with improved selectivity to detect tumor exosomes by recording the fluorescence intensity with λex/em = 557/580 nm. The standard curve for detecting tumor exosomes with the aptasensor was calculated as y = 371.7x + 66.17, ranging from 0.05 to 1 × 104 particles/µL, with R2 = 0.9737, and a detection limit of 5 × 102 particles/µL in solution. This method was successfully applied to clinical samples, and the results showed better performance in distinguishing prostate cancer patients and healthy samples than the traditional nanoparticle-tracking analysis (NTA) method. This rapid and accurate detection method for prostate cancer may aid in rapid clinical diagnosis. Integrating quickly TiO2-based isolation with sensitive and specific "on-off" detection of PCa exosomes.


Asunto(s)
Técnicas Biosensibles/métodos , Exosomas , Nanopartículas de Magnetita/química , Neoplasias de la Próstata/diagnóstico , Antígenos de Superficie/química , Aptámeros de Nucleótidos/química , Exosomas/química , Colorantes Fluorescentes/química , Glutamato Carboxipeptidasa II/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Masculino , Neoplasias de la Próstata/sangre , Rodaminas/química , Dióxido de Silicio/química , Espectrometría de Fluorescencia/métodos , Titanio/química , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
2.
Nucleic Acids Res ; 49(19): e111, 2021 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-34450653

RESUMEN

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.


Asunto(s)
Cartilla de ADN/química , VIH-1/química , Chaperonas Moleculares/química , Proteínas de la Nucleocápside/química , Péptidos/química , Albúmina Sérica Humana/química , Emparejamiento Base , Cartilla de ADN/metabolismo , Fluoresceínas/química , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , VIH-1/metabolismo , Humanos , Cinética , Técnicas Analíticas Microfluídicas , Chaperonas Moleculares/metabolismo , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/metabolismo , Péptidos/metabolismo , Albúmina Sérica Humana/metabolismo , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
3.
Amino Acids ; 53(7): 1033-1049, 2021 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-34032919

RESUMEN

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4-((4-(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.


Asunto(s)
Antineoplásicos/farmacología , Cationes/química , Péptidos de Penetración Celular/farmacología , Neoplasias/patología , Oligopéptidos/química , Péptidos/química , p-Dimetilaminoazobenceno/análogos & derivados , Antineoplásicos/química , Proliferación Celular , Péptidos de Penetración Celular/química , Humanos , Neoplasias/tratamiento farmacológico , Células Tumorales Cultivadas , p-Dimetilaminoazobenceno/química
4.
Angew Chem Int Ed Engl ; 60(13): 7333-7343, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33615660

RESUMEN

Live-cell delivery of a fully synthetic protein having selectivity towards a particular target is a promising approach with potential applications for basic research and therapeutics. Cell-penetrating peptides (CPPs) allow the cellular delivery of proteins but mostly result in endosomal entrapment, leading to lack of bioavailability. Herein, we report the design and synthesis of a CPP fused to 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL) to enhance cellular uptake of fluorescently labelled synthetic protein analogues in low micromolar concentration. The attachment of cyclic deca-arginine (cR10) modified with a single lysine linked to DABCYL to synthetic ubiquitin (Ub) and small ubiquitin-like modifier-2 (SUMO-2) scaffolds resulted in a threefold higher uptake efficacy in live cells compared to the unmodified cR10. We could also achieve cR10DABCYL-assisted delivery of Ub and a Ub variant (Ubv) based activity-based probes for functional studies of deubiquitinases in live cells.


Asunto(s)
Péptidos de Penetración Celular/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina/metabolismo , p-Dimetilaminoazobenceno/análogos & derivados , Línea Celular Tumoral , Péptidos de Penetración Celular/química , Fluorescencia , Humanos , Estructura Molecular , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/síntesis química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Ubiquitina/síntesis química , Ubiquitina/química , p-Dimetilaminoazobenceno/química , p-Dimetilaminoazobenceno/metabolismo
5.
Nat Commun ; 11(1): 784, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32034159

RESUMEN

Relatively robust dynamic covalent interactions have been employed extensively to mediate molecular self-assembly reactions; however, these assembly processes often do not converge to a thermodynamic equilibrium, instead yielding mixtures of kinetically-trapped species. Here, we report a dynamic covalent self-assembly process that mitigates kinetic trapping such that multiple unique oligomers bearing covalently coreactive pendant groups are able to undergo simultaneous, sequence-selective hybridization with their complementary strands to afford biomimetic, in-registry molecular ladders with covalent rungs. Analogous to the thermal cycling commonly employed for nucleic acid melting and annealing, this is achieved by raising and lowering the concentration of a multi-role reagent to effect quantitative dissociation and subsequently catalyze covalent bond rearrangement, affording selective assembly of the oligomeric sequences. The hybridization specificity afforded by this process further enabled information encoded in oligomers to be retrieved through selective hybridization with complementary, mass-labeled sequences.


Asunto(s)
Bioquímica/métodos , Peptoides/química , Aldehídos/química , Aminas/química , Fluoresceínas/química , Transferencia Resonante de Energía de Fluorescencia , Iminas/química , Cinética , Hibridación de Ácido Nucleico , Peptoides/síntesis química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
7.
Anal Biochem ; 585: 113400, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31437428

RESUMEN

In this short note we describe the conversion of the widely used fluorescence quenching azo-dyes DABCYL and HABA to fluorophores. The dyes were conjugated to the proteins RNase and human serum albumin (HSA) and subsequently reduced using sodium dithionite (Na2S2O4), thus forming amine-containing fluorophores. Since this chemical reaction can be applied to any azo-containing quencher compound, a great variety of substances can be readily obtained synthetically. This approach provides a promising tool in the use of fluorescence-based investigations of biomolecular interactions.


Asunto(s)
Compuestos Azo/química , Colorantes Fluorescentes/química , p-Dimetilaminoazobenceno/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Ditionita/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Estructura Molecular , Oxidación-Reducción , Unión Proteica , Ribonucleasas/química , Albúmina Sérica/química , Relación Estructura-Actividad , p-Dimetilaminoazobenceno/química
8.
Anal Chem ; 91(18): 11529-11536, 2019 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-31398009

RESUMEN

We report a tetrahedron-based DNAzyme probe (Tetra-ES) for intracellular miRNA detection. Two DNA tetrahedra (Tetra) were arranged at the different positions of the enzyme (E)/substrate (S) complex in a unique direction. A Na+-dependent DNAzme was designed to be initially locked to inhibit the activity of the DNAzyme. Fluorescence imaging and gel electrophoresis analyses demonstrated that the silenced DNAzyme could be specifically initiated by intracellular target miRNA. The activated DNAzyme repeatedly cleaved the substrates, allowing a controllable signal transduction and amplification effect. The combination of spatially controlled arrangement of DNA tetrahedra with the stimuli-responsive behavior of the locked DNAzyme improved cell permeability and desirable nuclease resistance. The Tetra-ES detector exhibited at least 10 times higher detection sensitivity (LOD of 16 pM) than that of the nonamplification molecular beacon counterpart and was capable of discriminating the miRNA target from the corresponding family members. The expression levels of target miRNA inside the cells of interest as well as different miRNAs inside the same type of cell lines were reliably screened utilizing the Tetra-ES detector. As an intracellular probe, Tetra-ES may provide valuable insight into developing a homogeneous DNA nanostructure-based controllable signal transduction strategy suitable for detection of miRNA and potential application to cancer diagnosis, prognosis, and therapeutics.


Asunto(s)
Sondas de ADN/química , ADN Catalítico/química , MicroARNs/análisis , Línea Celular Tumoral , Sondas de ADN/genética , Sondas de ADN/metabolismo , ADN Catalítico/genética , ADN Catalítico/metabolismo , Fluoresceínas/química , Colorantes Fluorescentes/química , Humanos , Límite de Detección , MicroARNs/química , MicroARNs/genética , Microscopía Fluorescente , Nanoestructuras/química , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Sodio/metabolismo , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
9.
Analyst ; 144(15): 4613-4621, 2019 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-31241068

RESUMEN

Lung cancer cells harbor various gene mutations in the mRNA sequence of the Epidermal Growth Factor Receptor (EGFR), especially the mutations of exon19del E746-A750, T790M, and L858R. This results in cancer progression and resistance to anticancer drugs (tyrosine kinase inhibitor; TKI). Therefore, the imaging analysis of EGFR mutations is required for the treatment planning for non-small cell lung cancers. This study focused on the imaging analysis of a single nucleotide substitute in EGFR mutated cancer cells. We developed three novel peptide nucleic acid (PNA)-DNA probes for recognizing and detecting the following three gene mutations in EGFR gene mutations. The PNA-DNA probes consist of fluorescein isothiocyanate (FITC) conjugated PNA as a detection probe and Dabcyl conjugated DNA as a quencher probe. The PNA-DNA probes were used to validate the feasibility for detecting three EGFR mutated sequences: exon19del E746-A750, T790M, and L858R. The three probes emitted fluorescent dose-dependent signals against three target DNA and RNA. Using the three PNA-DNA probes, we succeeded in distinguishing three kinds of lung-cancer cell lines (H1975, PC-9, and A549) which have different EGFR mutations by the fluorescence in situ hybridization (FISH) method.


Asunto(s)
Sondas de ADN/química , ADN/genética , Ácidos Nucleicos de Péptidos/química , ARN Mensajero/genética , Línea Celular Tumoral , Sondas de ADN/genética , Receptores ErbB/genética , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Humanos , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/genética , Hibridación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/genética , Mutación Puntual , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
10.
Carbohydr Polym ; 205: 385-391, 2019 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-30446119

RESUMEN

Heparanase, an endo-ß-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains and plays important roles in cellular growth and metastasis. Heparanase assays reported to-date are labor intensive, complex and/or expensive. A simpler assay is critically needed to understand the myriad roles of heparanase. We reasoned that fluorescent heparin could serve as an effective probe of heparanase levels. Following synthesis and screening, a heparin preparation labeled with DABCYL and EDANS was identified, which exhibited a characteristic increase in signal following cleavage by human heparanase. This work describes the synthesis of this heparin substrate, its kinetic and spectrofluorometric properties, optimization of the heparanase assay, use of the assay in inhibitor screening, and elucidation of the state of heparanase in different cell lines. Our FRET-based assay is much simpler and more robust than all assays reported in the literature as well as a commercially available kit.


Asunto(s)
Colorantes Fluorescentes/química , Glucuronidasa/química , Heparina/análogos & derivados , Heparina/química , Naftalenosulfonatos/química , p-Dimetilaminoazobenceno/análogos & derivados , Animales , Pruebas de Enzimas , Transferencia Resonante de Energía de Fluorescencia/métodos , Células HEK293 , Heparina/síntesis química , Humanos , Células MCF-7 , Naftalenosulfonatos/síntesis química , Células Sf9 , Spodoptera , p-Dimetilaminoazobenceno/síntesis química , p-Dimetilaminoazobenceno/química
11.
J Chromatogr A ; 1556: 21-28, 2018 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-29731293

RESUMEN

In the present research, an on-chip electromembrane extraction coupled with high performance liquid chromatography was developed for monitoring the trace levels of biogenic amines (BAs), including histamine, tryptamine, putrescine, cadaverine and spermidine in food samples. A porous polypropylene sheet membrane impregnated with an organic solvent was placed between the two parts of the chip device to separate the channels. Two platinum electrodes were mounted at the bottom of these channels, which were connected to a power supply, providing the electrical driving force for migration of ionized analytes from the sample solution through the porous sheet membrane into the acceptor phase. BAs were extracted from 2 mL aqueous sample solutions at neutral pH into 50 µL of acidified (HCl 90 mM) acceptor solution. Supported liquid membrane including NPOE containing 10% DEHP was used to ensure efficient extraction. Low voltage of 40 V was applied over the SLMs during extraction time. The influences of fundamental parameters affecting the transport of BAs were optimized. Under the optimized conditions, the relative standard deviations based on four replicate measurements were less than 8.0% and limit of detections were in range of 3.0-8.0 µg L-1. Finally, the method was successfully applied to determinate BAs in the food samples and satisfactory results (recovery > 95.6) were obtained.


Asunto(s)
Aminas Biogénicas/aislamiento & purificación , Cromatografía Líquida de Alta Presión/métodos , Electroquímica , Análisis de los Alimentos/métodos , Membranas Artificiales , Microfluídica/métodos , p-Dimetilaminoazobenceno/análogos & derivados , Aminas Biogénicas/análisis , Tampones (Química) , Electricidad , Electrodos , Reproducibilidad de los Resultados , Reología , Soluciones , Solventes/química , Temperatura , Factores de Tiempo , p-Dimetilaminoazobenceno/química
12.
Toxicol Lett ; 289: 75-85, 2018 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-29545173

RESUMEN

Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.


Asunto(s)
Cisteamina/antagonistas & inhibidores , Erupciones por Medicamentos/prevención & control , Drogas en Investigación/efectos adversos , Glutatión/antagonistas & inhibidores , Modelos Moleculares , Piel/efectos de los fármacos , Métodos Analíticos de la Preparación de la Muestra , Cromatografía Líquida de Alta Presión , Cisteamina/química , Evaluación Preclínica de Medicamentos/métodos , Drogas en Investigación/química , Glutatión/química , Humanos , Indicadores y Reactivos/química , Cinética , Fotometría , Curva ROC , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas , Espectrofotometría Ultravioleta , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
13.
Anal Chem ; 90(5): 3556-3562, 2018 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-29443497

RESUMEN

The development of well-designed nanoprobes for specific imaging of multiple biomarkers in renal cells will afford beneficial information related to the transmutation process of drug-induced kidney injury (DIKI). However, the most reported nanoprobes for DIKI detection were dependent on single-signal output and lack of kidney targeting. In this work, we reported a renal cell targeting and dual-signal nanoprobe by encapsulating Brite 670 and Dabcyl-KFFFDEVDK-FAM into a low molecular weight chitosan nanoparticle. Confocal fluorescence imaging results demonstrated that the nanoprobe could visualize the upregulation of hydroxyl radical in early stage and activation of caspase-3 in late stage of DIKI at both the renal cell and tissue level. In a mouse DIKI model, the positive time of 8 h using nanoprobe imaging was superior to that of 72 h for serum creatinine or blood urea nitrogen, 16 h for cystatin-C, and 24 h for kidney injury molecule-1 with conventional methods. These results demonstrated that the nanoprobe may be a promising tool for effective early prediction and discriminative imaging of DIKI.


Asunto(s)
Caspasa 3/análisis , Colorantes Fluorescentes/química , Radical Hidroxilo/análisis , Nanopartículas/química , Insuficiencia Renal/inducido químicamente , Insuficiencia Renal/diagnóstico por imagen , p-Dimetilaminoazobenceno/análogos & derivados , Animales , Línea Celular , Quitosano/química , Ratones , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Péptidos/química , Ratas , p-Dimetilaminoazobenceno/química
14.
Anal Chem ; 90(6): 3928-3935, 2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29465226

RESUMEN

Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search for endogenous kinases in human cellular extract.


Asunto(s)
Cisteína/análisis , Péptidos/química , Proteínas Quinasas/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Línea Celular , Humanos , Proteoma/química , Programas Informáticos , Flujo de Trabajo , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
15.
Cell Chem Biol ; 25(3): 301-308.e12, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29337186

RESUMEN

As resistance to antibiotics increases, the exploration of new targets and strategies to combat pathogenic bacteria becomes more urgent. Ideal protein targets are required for viability across many species, are unique to prokaryotes to limit effects on the host, and have robust assays to quantitate activity and identify inhibitors. Lipoprotein signal peptidase (Lsp) is a transmembrane aspartyl protease required for lipoprotein maturation and comprehensively fits these criteria. Here, we have developed the first in vitro high-throughput assay to monitor proteolysis by Lsp. We employed our high-throughput screen assay against 646,275 compounds to discover inhibitors of Lsp and synthesized a range of analogs to generate molecules with nanomolar half maximal inhibitory concentration values. Importantly, our inhibitors are effective in preventing the growth of E. coli cultures in the presence of outer-membrane permeabilizer PMBN and should facilitate development of antibacterial agents with a novel mechanism of action to treat antibiotic-resistant bacteria.


Asunto(s)
Antibacterianos/química , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Antibacterianos/farmacología , Ácido Aspártico Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Transferencia Resonante de Energía de Fluorescencia , Concentración 50 Inhibidora , Naftalenosulfonatos/química , Péptidos/química , Péptidos/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
16.
Biochemistry ; 56(41): 5550-5559, 2017 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-28945359

RESUMEN

DNA polymerases synthesize new DNA during DNA replication and repair, and their ability to do so faithfully is essential to maintaining genomic integrity. DNA polymerase ß (Pol ß) functions in base excision repair to fill in single-nucleotide gaps, and variants of Pol ß have been associated with cancer. Specifically, the E288K Pol ß variant has been found in colon tumors and has been shown to display sequence-specific mutator activity. To probe the mechanism that may underlie E288K's loss of fidelity, a fluorescence resonance energy transfer system that utilizes a fluorophore on the fingers domain of Pol ß and a quencher on the DNA substrate was employed. Our results show that E288K utilizes an overall mechanism similar to that of wild type (WT) Pol ß when incorporating correct dNTP. However, when inserting the correct dNTP, E288K exhibits a faster rate of closing of the fingers domain combined with a slower rate of nucleotide release compared to those of WT Pol ß. We also detect enzyme closure upon mixing with the incorrect dNTP for E288K but not WT Pol ß. Taken together, our results suggest that E288K Pol ß incorporates all dNTPs more readily than WT because of an inherent defect that results in rapid isomerization of dNTPs within its active site. Structural modeling implies that this inherent defect is due to interaction of E288K with DNA, resulting in a stable closed enzyme structure.


Asunto(s)
Neoplasias del Colon/enzimología , ADN Polimerasa beta/metabolismo , Reparación del ADN , Replicación del ADN , ADN/metabolismo , Modelos Moleculares , Mutación , Sustitución de Aminoácidos , Biocatálisis , Neoplasias del Colon/genética , ADN/química , ADN Polimerasa beta/química , ADN Polimerasa beta/genética , Estabilidad de Enzimas , Colorantes Fluorescentes/química , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Naftalenosulfonatos/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
17.
Chembiochem ; 18(16): 1568-1572, 2017 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-28586120

RESUMEN

Photodriven DNA strand displacement by using a 2',6'-dimethylazobenzene-tethered strand and poly(l-lysine)-graft-dextran (PLL-g-Dex) as a chaperone is reported. Rapid strand displacement was reversibly induced by UV and visible-light irradiation without any toehold portion. To further improve the method, the concentration of PLL-g-Dex and the number of equivalents of the photoresponsive strand were optimised. Optimally, 64 % strand displacement was reversibly induced by alternating UV and visible-light irradiation.


Asunto(s)
Compuestos Azo/efectos de la radiación , ADN/efectos de la radiación , Dextranos/química , Polilisina/análogos & derivados , Compuestos Azo/química , ADN/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Isomerismo , Nanotecnología , Hibridación de Ácido Nucleico , Polilisina/química , Temperatura de Transición , Rayos Ultravioleta , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
18.
J Hazard Mater ; 324(Pt B): 626-633, 2017 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-27887814

RESUMEN

Octyl-dimethyl-p-aminobenzoic acid (ODPABA), one of the most commonly used organic UV filters, can undergo considerable transformation in water when entering into the disinfection process. The impacts of bromide on degradation kinetics, formation and speciation of transformation products, regulated disinfection by-products (DBPs) as well as genotoxicity changes during ODPABA chlorination were investigated in this study. Results indicated that the reaction of ODPABA with chlorine followed pseudo-first-order and second-order kinetics. Adding bromide noticeably enhanced the degradation rate of ODPABA, but reduced the impact of chlorine dose. Four halogenated transformation products (Cl-ODPABA, Br-ODPABA, Cl-Br-ODPABA and Br2-ODPABA) were detected by LC-MS/MS. Mono-halogenated products were stable during 24-h chlorination, while di-halogenated products constantly increased. The total yields of trihalomethanes (THMs) and haloacetic acids (HAAs) were both low, but predominated by bromine substitution at high levels of bromide. In addition, SOS/umu tests showed that genotoxicity was generated after ODPABA chlorination, which was increased at least 1.5 times in the presence of bromine. Whereas, no significant genotoxicity variation was observed following bromide concentration change.


Asunto(s)
Bromuros/química , Desinfectantes/química , Mutágenos/química , Protectores Solares/química , Contaminantes Químicos del Agua/química , p-Dimetilaminoazobenceno/análogos & derivados , Desinfección , Halogenación , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Protectores Solares/toxicidad , Contaminantes Químicos del Agua/toxicidad , Purificación del Agua/métodos , p-Dimetilaminoazobenceno/química , p-Dimetilaminoazobenceno/toxicidad
19.
Sci Rep ; 6: 36006, 2016 10 31.
Artículo en Inglés | MEDLINE | ID: mdl-27796331

RESUMEN

DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases.


Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN Circular/química , Fluoresceína/química , Secuencia de Bases , Girasa de ADN/metabolismo , ADN Circular/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/metabolismo , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
20.
Bioconjug Chem ; 27(10): 2332-2336, 2016 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-27583637

RESUMEN

The ability of a molecular beacon to detect miR-132, a microRNA associated with the childhood cancer neuroblastoma, is reported in solution and within live cells. The stem-loop structure comprises a sequence complementary to miR-132, modified with a 6-FAM dye and dabcyl quencher on either end. In the absence of the target, self-binding occurs bringing the luminophore and quencher into close proximity, significantly decreasing the emission intensity. In the presence of miR-132, the signal is greatly enhanced, with a linear increase in intensity for mole ratios of beacon-to-target between 0.25 and 2.00. The structure differentiates between target and mismatched nucleic acid sequences, e.g., in the presence of a single-base mismatch, no increase in emission intensity beyond the background is observed. The stem-loop can be introduced into neuroblastoma cancer cells by electroporation, allowing miR-132 to be imaged within live cells. miR-132 appears to be localized within the nucleus of the cells, where its concentration is of the order of 1 µM. Significantly, transfection of the cells with a miR-132 mimic causes the emission intensity to more than double, demonstrating the sensitivity of the approach to changes in miR-132 concentration in live cells. This behavior opens up significant theranostic applications, such as the possibility of rapidly identifying retinoic acid resistant patients as well as providing a means to monitor therapeutic efficacy.


Asunto(s)
MicroARNs/análisis , Imagen Molecular/métodos , Neuroblastoma/genética , Línea Celular Tumoral , Fraccionamiento Químico/métodos , Colorantes Fluorescentes/química , Humanos , MicroARNs/genética , MicroARNs/aislamiento & purificación , Microscopía Confocal/métodos , Transfección , p-Dimetilaminoazobenceno/análogos & derivados , p-Dimetilaminoazobenceno/química
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