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1.
Food Chem ; 356: 129663, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33812184

RESUMO

A two-color fluorescent DNA tweezers was developed for ultrasensitive detection of Ochratoxin A (OTA) based on hairpin-locked aptamer and hybridization chain reaction (HCR) amplification strategy. OTA can bind with hairpin-locked aptamer and then trigger the HCR reaction to produce a long double-strand DNA. The side-chains of the long duplex can separately hybridize with the two locker sequences of DNA tweezer, causing the opening of DNA tweezer and the recovery of two-color fluorescent signals. It shows a good linear range from 0.02 to 0.8 ppb with limit of detection of 0.006 ppb for FAM and 0.014 ppb for Cy5, which is beyond the requirement of actual application. In addition, the two-color fluorescent strategy can greatly reduce the false positive rate. It shows excellent performance for detection of OTA in practical food sample.


Assuntos
DNA/química , Corantes Fluorescentes/química , Ocratoxinas/análise , Espectrometria de Fluorescência/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Cor , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Limite de Detecção , Hibridização de Ácido Nucleico , Ocratoxinas/metabolismo
2.
Nat Commun ; 12(1): 2366, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888692

RESUMO

Aptamers are single-stranded nucleic acid ligands that bind to target molecules with high affinity and specificity. They are typically discovered by searching large libraries for sequences with desirable binding properties. These libraries, however, are practically constrained to a fraction of the theoretical sequence space. Machine learning provides an opportunity to intelligently navigate this space to identify high-performing aptamers. Here, we propose an approach that employs particle display (PD) to partition a library of aptamers by affinity, and uses such data to train machine learning models to predict affinity in silico. Our model predicted high-affinity DNA aptamers from experimental candidates at a rate 11-fold higher than random perturbation and generated novel, high-affinity aptamers at a greater rate than observed by PD alone. Our approach also facilitated the design of truncated aptamers 70% shorter and with higher binding affinity (1.5 nM) than the best experimental candidate. This work demonstrates how combining machine learning and physical approaches can be used to expedite the discovery of better diagnostic and therapeutic agents.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Aprendizado de Máquina , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Simulação por Computador , Descoberta de Drogas/métodos , Biblioteca Gênica , Ligantes , Lipocalina-2/química , Lipocalina-2/genética , Lipocalina-2/metabolismo , Modelos Químicos , Ligação Proteica
3.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808496

RESUMO

Aptamers are short single-stranded DNA, RNA, or synthetic Xeno nucleic acids (XNA) molecules that can interact with corresponding targets with high affinity. Owing to their unique features, including low cost of production, easy chemical modification, high thermal stability, reproducibility, as well as low levels of immunogenicity and toxicity, aptamers can be used as an alternative to antibodies in diagnostics and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX), an experimental approach for aptamer screening, allows the selection and identification of in vitro aptamers with high affinity and specificity. However, the SELEX process is time consuming and characterization of the representative aptamer candidates from SELEX is rather laborious. Artificial intelligence (AI) could help to rapidly identify the potential aptamer candidates from a vast number of sequences. This review discusses the advancements of AI pipelines/methods, including structure-based and machine/deep learning-based methods, for predicting the binding ability of aptamers to targets. Structure-based methods are the most used in computer-aided drug design. For this part, we review the secondary and tertiary structure prediction methods for aptamers, molecular docking, as well as molecular dynamic simulation methods for aptamer-target binding. We also performed analysis to compare the accuracy of different secondary and tertiary structure prediction methods for aptamers. On the other hand, advanced machine-/deep-learning models have witnessed successes in predicting the binding abilities between targets and ligands in drug discovery and thus potentially offer a robust and accurate approach to predict the binding between aptamers and targets. The research utilizing machine-/deep-learning techniques for prediction of aptamer-target binding is limited currently. Therefore, perspectives for models, algorithms, and implementation strategies of machine/deep learning-based methods are discussed. This review could facilitate the development and application of high-throughput and less laborious in silico methods in aptamer selection and characterization.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Previsões/métodos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Inteligência Artificial/tendências , Simulação por Computador , Desenho Assistido por Computador/tendências , Ligantes , Simulação de Acoplamento Molecular , Ácidos Nucleicos/química , Reprodutibilidade dos Testes
4.
Molecules ; 26(5)2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800717

RESUMO

RNA aptamers are becoming increasingly attractive due to their superior properties. This review discusses the early stages of aptamer research, the main developments in this area, and the latest technologies being developed. The review also highlights the advantages of RNA aptamers in comparison to antibodies, considering the great potential of RNA aptamers and their applications in the near future. In addition, it is shown how RNA aptamers can form endless 3-D structures, giving rise to various structural and functional possibilities. Special attention is paid to the Mango, Spinach and Broccoli fluorescent RNA aptamers, and the advantages of split RNA aptamers are discussed. The review focuses on the importance of creating a platform for the synthesis of RNA nanoparticles in vivo and examines yeast, namely Saccharomyces cerevisiae, as a potential model organism for the production of RNA nanoparticles on a large scale.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Corantes Fluorescentes/química , Nanomedicina , Nanopartículas/administração & dosagem , Animais , Aptâmeros de Nucleotídeos/administração & dosagem , Brassica/metabolismo , Humanos , Mangifera/metabolismo , Nanopartículas/química , Saccharomyces cerevisiae/metabolismo , Spinacia oleracea/metabolismo
5.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-33673708

RESUMO

Nucleic acid aptamers are generally accepted as promising elements for the specific and high-affinity binding of various biomolecules. It has been shown for a number of aptamers that the complexes with several related proteins may possess a similar affinity. An outstanding example is the G-quadruplex DNA aptamer RHA0385, which binds to the hemagglutinins of various influenza A virus strains. These hemagglutinins have homologous tertiary structures but moderate-to-low amino acid sequence identities. Here, the experiment was inverted, targeting the same protein using a set of related, parallel G-quadruplexes. The 5'- and 3'-flanking sequences of RHA0385 were truncated to yield parallel G-quadruplex with three propeller loops that were 7, 1, and 1 nucleotides in length. Next, a set of minimal, parallel G-quadruplexes with three single-nucleotide loops was tested. These G-quadruplexes were characterized both structurally and functionally. All parallel G-quadruplexes had affinities for both recombinant hemagglutinin and influenza virions. In summary, the parallel G-quadruplex represents a minimal core structure with functional activity that binds influenza A hemagglutinin. The flanking sequences and loops represent additional features that can be used to modulate the affinity. Thus, the RHA0385-hemagglutinin complex serves as an excellent example of the hypothesis of a core structure that is decorated with additional recognizing elements capable of improving the binding properties of the aptamer.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Quadruplex G , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Animais , Aptâmeros de Nucleotídeos/química , Galinhas , Cricetinae , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Infecções por Orthomyxoviridae/virologia
6.
Food Chem ; 352: 129352, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691206

RESUMO

A ratiometric fluorescence sensor system is proposed for detecting bis(2-ethylhexyl) phthalate (DEHP) in pork, which is based on aptamer recognition with molybdenum disulfide quantum dots and cadmium telluride quantum dots (MoS2 QDs/CdTe-Apta). Two signals exist in the system, among which the response signal is transmitted by CdTe-Apta. The amide condensation between aptamers and CdTe QDs shortens the distance between CdTe QDs and DEHP, thus quenching the fluorescence of CdTe QDs, possibly through a photoinduced electron transfer mechanism. The MoS2 QDs deliver the self-calibration signal, and the fluorescence of MoS2 QDs remains almost constant when co-existing with DEHP. Linearity (R2 = 0.9536) was established for the DEHP concentration range 0.005-3.0 mg·L-1, with a limit of detection of 0.21 µg·L-1. The system was successfully applied in the determination of DEHP in pork. The system has potential for the quantitative determination of DEHP in practical applications.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Dietilexilftalato/análise , Carne de Porco/análise , Animais , Compostos de Cádmio/química , Dietilexilftalato/metabolismo , Dissulfetos/química , Molibdênio/química , Pontos Quânticos/química , Espectrometria de Fluorescência , Suínos , Telúrio/química
7.
Food Chem ; 352: 129330, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33657486

RESUMO

Pregnancy test strips are one of the most mature and widely used commercial lateral flow devices used to determine pregnancy. Being a simple and rapid detection method, human chorionic gonadotropin (hCG) was used with different aptamers (hCG-apt) as probes for the detection of metal ions, small organic molecules, and proteins. Quantitative detection of target analytes was achieved using a smartphone app and a portable device developed in our laboratory. The results showed detection ranges of 1 nM-1 µM, 0.1 nM-10 µM and 32 nM-500 nM for Pb2+, chloramphenicol, and ß-lactoglobulin, respectively, and the corresponding visual detection limits in dairy products were 5 nM, 1 nM and 50 nM, respectively. Based on these results, rapid detection of multiple analytes can be realized through aptamer modification, thereby broadening the application range of commercial lateral flow devices for analysis of food chemistry.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Laticínios/análise , Compostos Férricos/química , Análise de Alimentos/instrumentação , Grafite/química , Testes de Gravidez/instrumentação , Smartphone , Animais , Feminino , Ouro/química , Humanos , Limite de Detecção , Gravidez
8.
J Vis Exp ; (168)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33720114

RESUMO

Although small regulatory RNAs (sRNAs) are widespread among the bacterial domain of life, the functions of many of them remain poorly characterized notably due to the difficulty of identifying their mRNA targets. Here, we described a modified protocol of the MS2-Affinity Purification coupled with RNA Sequencing (MAPS) technology, aiming to reveal all RNA partners of a specific sRNA in vivo. Broadly, the MS2 aptamer is fused to the 5' extremity of the sRNA of interest. This construct is then expressed in vivo, allowing the MS2-sRNA to interact with its cellular partners. After bacterial harvesting, cells are mechanically lysed. The crude extract is loaded into an amylose-based chromatography column previously coated with the MS2 protein fused to the maltose binding protein. This enables the specific capture of MS2-sRNA and interacting RNAs. After elution, co-purified RNAs are identified by high-throughput RNA sequencing and subsequent bioinformatic analysis. The following protocol has been implemented in the Gram-positive human pathogen Staphylococcus aureus and is, in principle, transposable to any Gram-positive bacteria. To sum up, MAPS technology constitutes an efficient method to deeply explore the regulatory network of a particular sRNA, offering a snapshot of its whole targetome. However, it is important to keep in mind that putative targets identified by MAPS still need to be validated by complementary experimental approaches.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Cromatografia de Afinidade , Bactérias Gram-Positivas/genética , Análise de Sequência de RNA , Sequência de Bases , Tampões (Química) , Fracionamento Celular , Análise de Dados , Regulação Bacteriana da Expressão Gênica , Humanos , Plasmídeos/genética , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/genética , Reprodutibilidade dos Testes , Staphylococcus aureus/genética
9.
J Environ Sci Health B ; 56(4): 370-377, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33616003

RESUMO

This study aimed to develop an aptasensor for paraquat detection by gold nanoparticles. The specific aptamer for paraquat was selected by using the SELEX process via capillary electrophoresis. Sixty-three aptamer candidates were amplified by asymmetric PCR and screened for paraquat binding using gold nanoparticles (AuNPs). Aggregation of AuNPs was specifically induced by desorption of paraquat binding aptamers from the surface of AuNPs as a result of aptamer-target interaction leading to the color change from red to purple. Aptamer 77F with the following sequence: 5'-AGGCTTACACCTGAAAAGCGGCTTAATTTACACTACTGTAT-3' was selected as a highly specific aptamer for paraquat. The detection limit of paraquat was 0.267 µg/mL by colorimetry and 1.573 µg/mL by the quartz crystal microbalance (QCM) technique. This aptamer showed specificity for paraquat by colorimetry. Dimethyl phophite, diethyl phophite and O,O diethyl thiophosphate potassium salt did not react by colorimetry but, exhibited a weak nonspecific reaction by QCM. This is first time that an aptasensor was used for detection of paraquat based on QCM. However, the aptasensor based on the colorimetric method simply uses a generation of a signal that can be detected by the naked eye. This technique is rapid, low cost easy to use and suitable for on-site detection of herbicide residue.


Assuntos
Aptâmeros de Nucleotídeos/química , Colorimetria/métodos , Paraquat/análise , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Colorimetria/instrumentação , Eletroforese Capilar , Ouro/química , Herbicidas/análise , Limite de Detecção , Nanopartículas Metálicas/química , Paraquat/metabolismo , Reação em Cadeia da Polimerase , Técnicas de Microbalança de Cristal de Quartzo/métodos , Sensibilidade e Especificidade
10.
Food Chem ; 351: 129292, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33626465

RESUMO

The detection of carbendazim (CBZ) is important for food safety and human health. However, most current analytical methods require large instruments and highly trained operators. In order to solve this problem, herein, an innovative portable and quantitative photothermal assay platform relying on a thermometer readout for the detection of CBZ has been developed. Gold nanoparticles (AuNPs), which exhibit a strong distance-dependent photothermal effect under specific laser irradiation, were utilized as indicators. The CBZ aptamer was introduced to protect AuNPs from salt-mediated aggregation. When CBZ is present, the binding event between CBZ and aptamer leads to the loss of the aptamer protective effect on AuNPs, and AuNP aggregation occurs. Under 650-nm laser irradiation, the increase in temperature associated with an AuNP-dependent photothermal effect is highly related to the CBZ concentration. Having the advantages of user-friendliness, low cost, quick response, and portability, this method has great potential for on-site applications.


Assuntos
Benzimidazóis/análise , Carbamatos/análise , Análise de Alimentos/métodos , Fungicidas Industriais/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Brassica/química , Brassica/metabolismo , Citrus/química , Citrus/metabolismo , Análise de Alimentos/instrumentação , Frutas/química , Frutas/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Espectrofotometria Ultravioleta , Temperatura , Termômetros
11.
Anal Methods ; 13(10): 1302-1307, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33533761

RESUMO

Aptamers have many useful attributes including specific binding to molecular targets. After aptamers are identified, their target binding must be characterized. Fluorescence anisotropy (FA) is one technique that can be used to characterize affinity and to optimize aptamer-target interactions. Efforts to make FA assays more efficient by reducing assay volume and time from mixing to measurement may save time and resources by minimizing consumption of costly reagents. Here, we use thrombin and two thrombin-binding aptamers as a model system to show that plate-based FA experiments can be performed in volumes as low as 2 µL per well with 20 minute incubations with minimal loss in assay precision. We demonstrate that the aptamer-thrombin interaction is best modelled with the Hill equation, indicating cooperative binding. The miniaturization of this assay has implications in drug development, as well as in the efficiency of aptamer selection workflows by allowing for higher throughput aptamer analysis.


Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/metabolismo , Bioensaio , Polarização de Fluorescência , Ligação Proteica , Trombina
12.
J Phys Chem Lett ; 12(8): 2166-2171, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33629859

RESUMO

The ongoing outbreak of the coronavirus infection has killed more than 2 million people. Herein, we demonstrate that Rhodamine 6G (Rh-6G) dye conjugated DNA aptamer-attached gold nanostar (GNS)-based distance-dependent nanoparticle surface energy transfer (NSET) spectroscopy has the capability of rapid diagnosis of specific SARS-CoV-2 spike recombinant antigen or SARS-CoV-2 spike protein pseudotyped baculovirus within 10 min. Because Rh-6G-attached single-stand DNA aptamer wrapped the GNS, 99% dye fluorescence was quenched because of the NSET process. In the presence of spike antigen or virus, the fluorescence signal persists because of the aptamer-spike protein binding. Specifically, the limit of detection for the NSET assay has been determined to be 130 fg/mL for antigen and 8 particles/mL for virus. Finally, we have demonstrated that DNA aptamer-attached GNSs can stop virus infection by blocking the angiotensin-converting enzyme 2 (ACE2) receptor binding capability and destroying the lipid membrane of the virus.


Assuntos
Antígenos Virais/análise , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , COVID-19/diagnóstico , Ouro/química , Nanopartículas Metálicas/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/análise , Enzima de Conversão de Angiotensina 2/metabolismo , Antígenos Virais/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Teste para COVID-19/métodos , Transferência de Energia , Humanos , Limite de Detecção , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Int J Mol Sci ; 22(3)2021 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-33498970

RESUMO

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, ß-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5' or 3' end or at both ends. The TT-tail at the 5' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.


Assuntos
Alérgenos , Antígenos de Plantas/química , Aptâmeros de Nucleotídeos/química , Quadruplex G , Globulinas/química , Proteínas de Armazenamento de Sementes/química , Proteínas de Soja/química , Antígenos de Plantas/imunologia , Aptâmeros de Nucleotídeos/metabolismo , Globulinas/imunologia , Estrutura Molecular , Conformação de Ácido Nucleico , Proteínas de Armazenamento de Sementes/imunologia , Proteínas de Soja/imunologia
14.
Nucleic Acids Res ; 49(2): 1065-1074, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398328

RESUMO

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA Nucleotidilexotransferase/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Aptâmeros de Nucleotídeos/biossíntese , Aptâmeros de Nucleotídeos/isolamento & purificação , Sítios de Ligação , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Lactoferrina/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Ligação Proteica , Especificidade por Substrato , Trombina/metabolismo , Recombinação V(D)J
15.
Food Chem ; 347: 128988, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33465686

RESUMO

A label-free colorimetric method based on exonuclease I (Exo I)-assisted signal amplification with protamine as a medium was developed for analysis of kanamycin. In this study, a double-stranded DNA (dsDNA) probe was tailored by manipulating an aptamer and its complementary DNA (cDNA) ensuring detection of target with high selectivity and excellent sensitivity. Herein, protamine could not only combine with negatively charged gold nanoparticles but also interaction with polyanion DNA. Upon addition of target kanamycin, the target-aptamer complex was formed and the cDNA was released. Thus, both aptamer and cDNA could be digested by Exo I, and the captured kanamycin was liberated for triggering target recycling and signal amplification. Under optimized conditions, the proposed colorimetric method realized a low detection limit of 2.8 × 10-14 M along with a wide linear range plus excellent selectivity. Our strategy exhibited enormous potentials for fabricate various kinds of biosensors based on target-induced aptamer configuration changes.


Assuntos
Colorimetria/métodos , Exodesoxirribonucleases/metabolismo , Canamicina/análise , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Sondas de DNA/química , Sondas de DNA/metabolismo , DNA Complementar/química , DNA Complementar/metabolismo , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Amplificação de Ácido Nucleico
16.
Food Chem ; 348: 129128, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-33516992

RESUMO

A novel colorimetric aptasensor based on unmodified gold nanoparticle (AuNPs) and single-strand DNA (ssDNA) aptamer was developed for the rapid and sensitive detection of T-2 toxin. In the absence of T-2, the AuNPs were wrapped by the aptamer to avoid the salt-induced aggregation and the solution remains red. In the presence of T-2, the aptamer was bound with T-2 and released from the surface of AuNPs, resulting in the aggregation of AuNPs under proper salt solution and the color change from red to purple-blue. The aptasensor exhibited a high sensitivity and selectivity for the detection of T-2. The range of linearity and detection limit were 0.1 ng/mL-5000 ng/mL (0.21435 nM-10717.5 nM) and 57.8 pg/mL (0.124 nM), respectively. The aptasensor developed here was applicable to assay T-2 in wheat and corn samples. These results implied that the colorimetric aptasensor was potentially useful in food detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Colorimetria/métodos , DNA de Cadeia Simples/química , Ouro/química , Nanopartículas Metálicas/química , Toxina T-2/análise , Aptâmeros de Nucleotídeos/metabolismo , Limite de Detecção , Triticum/metabolismo , Zea mays/metabolismo
17.
RNA ; 27(4): 390-402, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33483368

RESUMO

G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base-pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated by loop nucleotides). Such a sequence restriction, and the stereochemical constraints inherent to RNA (arising, in particular, from the presence of the 2'-OH), dictate relatively simple RNA G4 structures. Recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers have revealed RNA G4 structures of unprecedented complexity. Structures of the Sc1 aptamer that binds an RGG peptide from the Fragile-X mental retardation protein, various fluorescence turn-on aptamers (Corn, Mango, and Spinach), and the spiegelmer that binds the complement protein C5a, in particular, reveal complexity hitherto unsuspected in RNA G4s, including nucleotides in syn conformation, locally inverted strand polarity, and nucleotide quartets that are not all-G. Common to these new structures, the sequences folding into G4s do not conform to the requirement that guanine stacks arise from consecutive (contiguous in sequence) nucleotides. This review highlights how emancipation from this constraint drastically expands the structural possibilities of RNA G-quadruplexes.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Guanina/química , RNA/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Complemento C5a/química , Complemento C5a/genética , Complemento C5a/metabolismo , Corantes Fluorescentes/química , Proteína do X Frágil de Retardo Mental/química , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Guanina/metabolismo , Humanos , Ligação Proteica , RNA/genética , RNA/metabolismo , Estereoisomerismo
18.
Eur J Pharmacol ; 894: 173814, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33352182

RESUMO

Pancreatic cancer is a high degree malignant tumor which makes its diagnosis and treatment highly critical. The effect of conventional chemotherapy for pancreatic cancer is quite poor due to the low accumulation of the chemotherapeutic drugs at the tumor site. Therefore, enhancing the targeting efficiency and accumulation of the drug carrier at tumor site with subsequent release of drug within the effective time period is one of the key factors for successful targeted chemotherapy of pancreatic cancer. Our previous studies have demonstrated that aptamer can be a valid targeting moiety to guide the chemotherapeutic drug doxorubicin (DOX) to accumulate at the tumor tissue. Herein, the present study aims to further investigate the targeting efficiency as well as therapeutic efficacy of the drug delivery system comprised of aptamer-modified polymeric nano drug carrier encapsulated with DOX (DOX@PCL-b-PEO-Aptamer micelles). The in vitro cytotoxicity studies and laser confocal microscopy indicated that DOX@PCL-b-PEO-Aptamer micelles exhibited enhanced targeting and cytotoxic efficacy towards human pancreatic cancer cells (Panc-1 cells) as compared to free DOX and DOX-loaded PCL-b-PEO-NH2 micelles (DOX@PCL-b-PEO-NH2 micelles). Furthermore, the aptamer-decorated drug delivery system exhibited better tumor penetration into the three-dimensional (3D) spheroid of Panc-1 cells with successful release of DOX as compared to the drug delivery system without aptamer modification. Overall, this study suggests that the aptamer-modified polymeric micelles could be effectively employed for the targeted delivery of anticancer drug to treat pancreatic cancer in near future.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Polímeros/metabolismo , Esferoides Celulares/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Células HEK293 , Humanos , Micelas , Polímeros/química
19.
Food Chem ; 339: 128059, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33152864

RESUMO

A colorimetric aptasensing strategy for detection of kanamycin was designed based on aptamer biorecognition and signal amplification assisted by nicking enzyme. The aptamer of kanamycin was designed to be contained in the metastable state hairpin DNA. The target DNA as recycling DNA was located in the loop of hairpin DNA. The presence of kanamycin stimulates the continuous actions, including specific recognition of the aptamer to kanamycin, the hybridization between target DNA and signal probe, the cleavage function of nicking enzyme. The actions induced accumulation of numerous free short sequences modified by platinum nanoparticles (PtNPs), which can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 to produce a colorimetric response. The aptasensor exhibited good selectivity and sensitivity for kanamycin in milk with a detection limit as low as 0.2 pg·mL-1. In addition, the proposed assay is potentially to be extended for other antibiotics detection in foods by adapting the corresponding aptamer sequence.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Desoxirribonuclease I/metabolismo , Sequências Repetidas Invertidas , Canamicina/análise , Leite/química , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Benzidinas/química , Sondas de DNA/química , Contaminação de Alimentos/análise , Peróxido de Hidrogênio/química , Canamicina/metabolismo , Limite de Detecção , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Platina/química
20.
Food Chem ; 340: 128181, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33032145

RESUMO

17ß-estradiol (E2) residues could enrich in organisms via food chain and lead to harmful biological effects for human body. To ascertain the binding domain of original E2 aptamer (E00) with long-sequence (76-mer), we developed novel truncated aptamers from E00, through rationally designed truncation by intercepting a single ring or a combination of rings (containing hairpin loop, interior loop or multiloop) at different sites and retaining appropriate double helix regions. Through comparison, 15-mer E09 presented improved affinity and higher specificity, indicating the hairpin loop near to 3' end of E00 served on the binding domain to E2. E09 was used for gold nanoparticles (AuNPs)-based colorimetric determination of E2, achieved the detection limit of 0.02 µg/mL. The truncated aptamer (only 15-mer) first proposed in this study has great application potential in E2 determination, and this work provides proof-of-concept study for truncation of other long-sequence aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/métodos , Colorimetria , Estradiol/análise , Ouro/química , Nanopartículas Metálicas/química , Estradiol/metabolismo , Humanos
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