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2.
Anal Chim Acta ; 1078: 176-181, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358217

RESUMO

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.


Assuntos
DNA de Cadeia Simples/química , MicroRNAs/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , Portadores de Fármacos/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
3.
Anal Chim Acta ; 1078: 42-52, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358227

RESUMO

Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN)63-/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 µA. cm-2, wide linear range of 1 nM-13.83 µM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Hemoglobina A Glicada/análise , Grafite/química , Nanopartículas Metálicas/química , Papel , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/instrumentação , DNA/química , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ouro/química , Humanos , Limite de Detecção , Nanocompostos/química , Reprodutibilidade dos Testes
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 533-539, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292058

RESUMO

Objective To screen aptamers that specifically bind to adhesin HpaA from Helicobacter pylori (H. pylori) by systematic evolution of ligands by exponential enrichment (SELEX) and identify the binding properties of aptamers. Methods The prokaryotic expression recombinant plasmid pET28a/HpaA was constructed and the HpaA protein was expressed and purified with IPTG. With the recombinant HpaA protein as target, we screened aptamers with high affinity and specificity binding force by SELEX. The binding force between aptamers and H. pylori in vitro and the performance of aptamers in H. pylori detection from the biopsy of gastric mucosa were examined using the aptamers we had screened. Results We extracted genome from H. pylori ATCC26695 strains and amplified 699 bp HpaA gene using PCR. The recombinant plasmid pET28a/HpaA was constructed successfully. The recombinant HpaA was expressed and purified up to 98% as target for aptamer screening. The six highest affinity aptamers were obtained and named HA1 to HA6 through 10-round positive screening and five-round negative screening by SELEX. The full-length aptamer HA6 and the core sequence of HA6 showed highest affinity and specificity in H. pylori detection in vitro. In view of this, the FAM-labelled aptamer HA6 was used to detect H. pylori in gastric mucosa from 166 patients. The aptamer HA6 showed a higher detection rate (94.58%) than URT (87.95%) in the same batch of clinical samples. Conclusion The aptamers that specifically bind to HpaA may be applied for the detection of H. pylori in gastric mucosa as a novel method.


Assuntos
Adesinas Bacterianas/genética , Aptâmeros de Nucleotídeos/genética , Mucosa Gástrica/microbiologia , Helicobacter pylori/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética
6.
Anal Chim Acta ; 1076: 55-63, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203964

RESUMO

In this work, an implantable and minimally invasive micro-aptasensor for adenosine monitoring in vivo, based on flexible integrated electrodes, was developed. Firstly the sensor was made by the modification of a needle-type electrode with reduced graphene oxide and gold nanoclusters (rGO-AuNCs) using two-step electrodeposition. Secondly Sulfhydryl-terminated capture probe (ssDNA1) was immobilized on rGO-AuNCs modified electrode surface by self-assembly, and then it was hybridized with adenosine aptamer (ssDNA2). Lastly methylene blue (MB) as an electrochemical indicator was adsorbed on the aptamer through specific interaction of MB with guanine base. The peak current of MB decreased linearly with increasing adenosine concentration due to the formation of aptamer-adenosine complex and displacement of the aptamer from the modified electrode surface. The sensor showed a low detection limit of 0.1 nM with signal-to-noise ratio equal to 3 as well as a wide linear response range (0.1 nM-1 mM) in vitro. Also, a high selectivity was demonstrated for adenosine in relation to uridine, guanosine, and cytidine. Experiments in vivo demonstrated fast responses for a range of adenosine concentrations. This work demonstrates a promising path for implantable devices for the determination of biomolecules in vivo, thus allowing for health tests, detection of infectious diseases, and other medical conditions.


Assuntos
Adenosina/análise , Aptâmeros de Nucleotídeos/química , DNA/química , Animais , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Grafite/química , Indicadores e Reagentes/química , Limite de Detecção , Masculino , Nanopartículas Metálicas/química , Azul de Metileno/química , Hibridização de Ácido Nucleico , Ratos Wistar
7.
Anal Chim Acta ; 1075: 128-136, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196418

RESUMO

A fluorescent detection of Staphylococcus aureus (S. aureus) is established based on a finely designed functional chimera sequence, a molecular beacon (MB), and strand displacement target recycling. The chimera sequence, which consists of the aptamer sequence of S. aureus and the complementary sequence of MB, can form a hairpin structure due to the existence of intramolecular complementary regions. When S. aureus is present, it binds to the aptamer region of the chimera, opens the hairpin and unlocks the complementary sequence of MB. Subsequently, the MB is opened and intensive fluorescence signal is restored. To increase the sensitivity of the detection, signal amplification is achieved through strand displacement-based target recycling. With the catalysis of Nb. Bpu10I nicking endonuclease and Bsm DNA polymerase, the MB sequence can be cleaved and then elongated to form a complete duplex with the chimera, during which S. aureus is displaced from the chimera and proceeded to the next round of the reaction. This assay displays a linear correlation between the fluorescence intensity and the logarithm of the concentration of S. aureus within a broad concentration range from 80 CFU/mL to 8 × 106 CFU/mL. The detection limit of 39 CFU/mL can be derived. The assay was applied to detect S. aureus in different water samples, and satisfactory recovery and repeatability were achieved. Hence the designed chimera sequence and established assay have potential application in environmental pollution monitoring.


Assuntos
Aptâmeros de Nucleotídeos/química , DNA Bacteriano/análise , Staphylococcus aureus/isolamento & purificação , Aptâmeros de Nucleotídeos/genética , Técnicas de Tipagem Bacteriana/métodos , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease I/química , Água Potável/microbiologia , Monitoramento Ambiental/métodos , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Lagos/microbiologia , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Tanques/microbiologia , Espectrometria de Fluorescência , Staphylococcus aureus/genética
8.
Nat Commun ; 10(1): 2829, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249296

RESUMO

Extracellular vesicles (EVs) are involved in the regulation of cell physiological activity and the reconstruction of extracellular environment. Matrix vesicles (MVs) are a type of EVs released by bone-related functional cells, and they participate in the regulation of cell mineralization. Here, we report bioinspired MVs embedded with black phosphorus (BP) and functionalized with cell-specific aptamer (denoted as Apt-bioinspired MVs) for stimulating biomineralization. The aptamer can direct bioinspired MVs to targeted cells, and the increasing concentration of inorganic phosphate originating from BP can facilitate cell biomineralization. The photothermal effect of the Apt-bioinspired MVs can also promote the biomineralization process by stimulating the upregulated expression of heat shock proteins and alkaline phosphatase. In addition, the Apt-bioinspired MVs display outstanding bone regeneration performance. Our strategy provides a method for designing bionic tools to study the mechanisms of biological processes and advance the development of medical engineering.


Assuntos
Vesículas Extracelulares/metabolismo , Fósforo/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Biomineralização , Osso e Ossos/química , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Vesículas Extracelulares/química , Feminino , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/química , Osteoblastos/metabolismo , Fosfatos/metabolismo , Fósforo/química , Ratos
9.
Anal Chim Acta ; 1074: 142-149, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159934

RESUMO

A simple proximity hybridization-induced on particle DNA walker was designed for ultrasensitive detection of proteins, for example platelet-derived growth factor (PDGF-BB) secreted by cancer cells, in which the DNA walker was activated by specific target binding and powered by an enzymatic cleavage to produce amplified signal. High-density FAM-labeled hairpin oligonucleotides (FAM-DNA1) were functionalized on AuNPs to construct three-dimensional (3D) DNA tracks. The specific binding of PDGF-BB with two aptamer probes (DNA3 and DNA4) led to the proximity hybridization-induced DNA displacement and the free of DNA walker (DNA2) to perform movement on the 3D tracks by an enzymatic cleavage, resulting in the release of massive FAM-DNA1 fragments from the AuNPs and the generation of fluorescent signal. This DNA walker based sensing strategy could detect PDGF-BB in a concentration range of 4 orders of magnitude with a detection limit down to sub-pM level. The practical applicability of the assay was demonstrated by detecting PDGF-BB secreted from MCF-7 cells with satisfactory results. The proposed DNA walker based assay could conveniently detect PDGF-BB with high sensitivity and good accuracy, along with the good extensibility of the assay, showing promise for practical diagnosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Becaplermina/análise , DNA/química , Trombina/análise , Aptâmeros de Nucleotídeos/genética , Bioensaio/métodos , DNA/genética , Ouro/química , Humanos , Limite de Detecção , Células MCF-7 , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico
10.
Comput Biol Chem ; 80: 452-462, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31170561

RESUMO

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Reativadores da Colinesterase/metabolismo , Paraoxon/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Reativadores da Colinesterase/química , Desenho de Drogas , Humanos , Ligações de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Paraoxon/química , Ligação Proteica , Eletricidade Estática
11.
Analyst ; 144(11): 3649-3658, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31074470

RESUMO

Serious healthcare concerns have been raised on the issue of antibiotic residues after overuse, especially by accumulation in the human body through food webs. Here, we report a methodological development for sensitive detection of antibiotics with aptamer conformation cooperated enzyme-assisted SERS (ACCESS) technology. We design and integrate a set of nucleic acid oligos, realizing specific recognition of chloramphenicol (CAP) and efficient exonuclease III-assisted DNA amplification. It features a "signal-on" analysis of CAP with the limit of detection (15 fM), the lowest concentration detectable in the literature. Our method exhibits a high selectivity on the target analyte, free of interference of other potential antibiotic contaminants. The ACCESS assay promises an ultrasensitive and specific detection tool for trace amounts of antibiotic residues in samples of our daily life.


Assuntos
Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Cloranfenicol/análise , Sondas de DNA/química , DNA/química , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/genética , Exodesoxirribonucleases/química , Contaminação de Alimentos/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Silício/química , Análise Espectral Raman/métodos , Poluentes Químicos da Água/análise
12.
Analyst ; 144(12): 3716-3720, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31134993

RESUMO

A photothermal immune-imaging assay was innovatively designed for the visual quantitative detection of cancer biomarkers by coupling CuxS nanocrystals with a portable infrared thermal imager on a smartphone. The rolling circle amplification (RCA) technique was used for the formation of a CuxS nanocrystal concatemer, thus opening up new territories in immunoassay development.


Assuntos
Biomarcadores Tumorais/análise , Imunoensaio/métodos , Nanocompostos/química , Antígeno Prostático Específico/análise , Smartphone , Anticorpos/imunologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Cobre/química , DNA/química , DNA/genética , Humanos , Imunoensaio/instrumentação , Raios Infravermelhos , Masculino , Nanocompostos/efeitos da radiação , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Antígeno Prostático Específico/imunologia , Sulfetos/química , Temperatura Ambiente
13.
Anal Sci ; 35(5): 585-588, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31080213

RESUMO

Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library. After only three selection rounds, we successfully enriched the modified aptamers and they showed slower off-rate than reported aptamers, suggesting MACE-SELEX is a promising approach for rapid identification of modified aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/isolamento & purificação , DNA/química , Eletroforese Capilar/métodos , Microesferas , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/genética , DNA/genética , Biblioteca Gênica , Humanos , Indóis/farmacologia , Trombina/antagonistas & inibidores
14.
Analyst ; 144(12): 3800-3806, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31116196

RESUMO

In typical photoelectrochemical (PEC) biosensing assays, electrodes are generally modified with photoactive probes and/or target recognition probes, which makes the processes complicated, time-consuming, and difficult to achieve excellent reproducibility. Hence, to overcome such shortcomings, we propose here an immobilization-free and label-free PEC aptasensor using solution-phase methylene blue (MB) as the PEC signal probe. Based on the unique T-Hg2+-T base pairs, and the diffusivity difference between free MB molecules and the MB/G-quadruplex composite towards the ITO electrode surface with negative charge, the "signal-off" approach for Hg2+ detection is developed. In the presence of target Hg2+, via the T-Hg2+-T bond formation, the two sticky ends of the hairpin DNA probe form a rigid duplex stem, which triggers the exonuclease III-facilitated target cycling amplification, and the formation of multiple G-quadruplexes. Upon the intercalation of MB in G-quadruplexes, significantly decreased photocurrent is obtained owing to the increased electrostatic repulsion between the MB/G-quadruplex composite and the ITO electrode. Therefore, highly sensitive and ultrasensitive Hg2+ determination is achieved, with a low detection limit of 1.2 pM, well below the maximum allowable Hg2+ level in drinking water defined by the WHO, China's Ministry of Health, and the US EPA. Due to the avoidance of sophisticated electrode modification and recognition probe immobilization processes, as well as an expensive labeling procedure, the PEC aptasensor proposed here demonstrates the advantages of simplicity, good reproducibility, rapidness and low cost.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Exodesoxirribonucleases/química , Mercúrio/análise , Azul de Metileno/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , DNA/química , DNA/genética , Sondas de DNA/genética , Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Quadruplex G , Substâncias Intercalantes/química , Substâncias Intercalantes/efeitos da radiação , Sequências Repetidas Invertidas , Lagos/análise , Limite de Detecção , Mercúrio/química , Azul de Metileno/efeitos da radiação , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Timina/química , Compostos de Estanho/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química
15.
Talanta ; 201: 52-57, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122460

RESUMO

More and more attention about food safety leads to a research hotspot to develop new detection methods for food contaminant. To address the problems of serious interference and low sensitivity, a chemiluminescent aptasensor for the detection of aflatoxin B1(AFB1) in food was developed in this paper. It is based on horseradish peroxidase (HRP) catalyze the luminol chemiluminescence reaction. The hybridization chain reaction (HCR) signal amplification strategy has been used to improve the detection sensitivity. Magnetic separation could further reduce background signal obviously at the same time. AFB1 as a model of analyte to test the capability of our developed assay system. Under the optimal experimental conditions, CL intensity showed a good linear correlation with the concentrations of AFB1 ranging from 0.5 to 40 ng mL-1. The limit of detection was estimated 0.2 ng mL-1 based on 3 times of the signal-to-noise ratio which is lower than those of the previously reported sensors. It could be used to detect AFB1 content in real samples, such as peanuts and milk which were purchased in local supermarket. The results proved that the sensing system has good anti-interference and selectivity. In all, it has potential for practical application in food safety field.


Assuntos
Aflatoxina B1/análise , Aptâmeros de Nucleotídeos/química , Arachis/química , Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Leite/química , Animais , Aptâmeros de Nucleotídeos/genética , Armoracia/enzimologia , DNA/química , DNA/genética , Peroxidase do Rábano Silvestre/química , Peróxido de Hidrogênio/química , Limite de Detecção , Luminescência , Medições Luminescentes , Luminol/química , Hibridização de Ácido Nucleico , Oxirredução
16.
Nat Commun ; 10(1): 2127, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31073154

RESUMO

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.


Assuntos
Aptâmeros de Nucleotídeos/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA Guia/genética , DNA/genética , Ligantes
17.
Analyst ; 144(10): 3389-3397, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-30990481

RESUMO

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Nanoestruturas/química , Ocratoxinas/análise , Aptâmeros de Nucleotídeos/genética , Fagos Bacilares/enzimologia , Bacteriófago T4/enzimologia , Sequência de Bases , DNA/genética , DNA Ligases/química , DNA Polimerase Dirigida por DNA/química , Desoxirribonuclease IV (Fago T4-Induzido)/química , Fluorescência , Corantes Fluorescentes/química , Contaminação de Alimentos/análise , Quadruplex G , Sequências Repetidas Invertidas , Limite de Detecção , Mesoporfirinas/química , Técnicas de Amplificação de Ácido Nucleico , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Ocratoxinas/química , Espectrometria de Fluorescência/métodos , Proteínas Virais/química , Vinho/análise
18.
Lab Chip ; 19(9): 1676-1685, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30942226

RESUMO

Although cardiovascular diseases such as heart failure (HF) affect 30 million people globally, the early detection of HF has, until recently, been difficult and prone to misdiagnoses. Monitoring the circulatory levels of a relatively new biomarker, the N-terminal prohormone of a B-type natriuretic peptide, could be used for early risk evaluation of HF. Therefore, we developed a pneumatically-driven, automatic integrated microfluidic platform equipped with micromixers, micropumps, and microvalves for the simultaneous detection of NT-proBNP in up to six clinical samples within 25 min by using a novel aptamer-based sandwich assay, and the limit of detection was only 1.53 pg mL-1; given that the chip is 64% more compact than those developed in our prior works and requires only 5 µL of sample input, it may serve as a promising tool for early diagnosis of HF.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , Dispositivos Lab-On-A-Chip , Peptídeo Natriurético Encefálico/análise , Fragmentos de Peptídeos/análise , Integração de Sistemas , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Calibragem , Desenho de Equipamento , Humanos , Limite de Detecção , Peptídeo Natriurético Encefálico/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Tempo
19.
Comput Biol Chem ; 80: 168-176, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965174

RESUMO

The alarm is rang for friendly fire; Saccharomyces cerevisiae (S. cerevisiae) newfound as a fungal pathogen with an individual feature. S. cerevisiae has food safety and is not capable of producing infection but, when the host defenses are weakened, there is room for opportunistic S. cerevisiae strains to cause a health issues. Fungal diseases are challenging to treat because, unlike bacteria, the fungal are eukaryotes. Antibiotics only target prokaryotic cells, whereas compounds that kill fungi also harm the mammalian host. Small differences between mammalian and fungal cells regarding genes and proteins sequence and function make finding a drug target more challenging. Recently, Chitin synthase has been considered as a promising target for antifungal drug development as it is absent in mammals. In S. cerevisiae, CHS3, a class IV chitin synthase, produces 90% of the chitin and essential for cell growth. CHS3 from the trans-Golgi network to the plasma membrane requires assembly of the exomer complex (including proteins cargo such as CHS5, CHS6, Bach1, and Arf1). In this work, we performed SELEX (Systematic Evolution of Ligands by EXponential enrichment) as high throughput virtual screening of the RCSB data bank to find an aptamer as potential inhibit of the class IV chitin synthase of S. cerevisiae. Among all the candidates, G-rich VEGF (GVEGF) aptamer (PDB code: 2M53) containing locked sugar parts was observed as potential inhibitor of the assembly of CHS5-CHS6 exomer complex a subsequently block the chitin biosynthesis pathway as an effective anti-fungal. It was suggested from the simulation that an assembly of exomer core should begin CHS5-CHS6, not from CHS5-Bach1. It is notable that secondary structures of CHS6 and Bach1 was observed very similar, but they have only 25% identity at the amino acid sequence that exhibited different features in exomer assembly.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Quitina Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Proteínas Adaptadoras de Transporte Vesicular/química , Sequência de Aminoácidos , Antifúngicos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Quitina Sintase/química , Quadruplex G , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência
20.
Nat Chem Biol ; 15(5): 472-479, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30992561

RESUMO

Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Aptâmeros de Nucleotídeos/genética , Mutação , Conformação de Ácido Nucleico
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