RESUMEN
Nucleic acid aptamers specific to S-protein and its receptor binding domain (RBD) of SARS-CoV-2 (severe acute respiratory syndrome-related coronavirus 2) virions are of high interest as potential inhibitors of viral infection and recognizing elements in biosensors. Development of specific therapy and biosensors is complicated by an emergence of new viral strains bearing amino acid substitutions and probable differences in glycosylation sites. Here, we studied affinity of a set of aptamers to two Wuhan-type RBD of S-protein expressed in Chinese hamster ovary cell line and Pichia pastoris that differ in glycosylation patterns. The expression system for the RBD protein has significant effects, both on values of dissociation constants and relative efficacy of the aptamer binding. We propose glycosylation of the RBD as the main force for observed differences. Moreover, affinity of a several aptamers was affected by a site of biotinylation. Thus, the robustness of modified aptamers toward new virus variants should be carefully tested.
Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Ácidos Nucleicos Inmovilizados/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetulus , Glicosilación , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Saccharomycetales/genéticaRESUMEN
Liposomal spherical nucleic acids (LSNAs) modified with polyethylene glycol (PEG) units are studied in an attempt to understand how the circulation time and biodistribution of the constructs can be manipulated. Specifically, the effect of (1) PEG molecular weight, (2) PEG shell stability, and (3) PEG modification method (PEG in both the core and shell versus PEG in the shell only) on LSNA blood circulation, biodistribution, and in vivo cell internalization in a syngeneic, orthotopic triple-negative breast cancer mouse model is studied. Generally, high PEG molecular weight extends blood circulation lifetime, and a more lipophilic anchor stabilizes the PEG shell and improves circulation and tumor accumulation but at the cost of cell uptake efficiency. The PEGylation strategy has a minor effect on in vitro properties of LSNAs but significantly alters in vivo cell uptake. For example, surface-only PEG in one design contributed to higher in vivo cell internalization than its counterpart with PEG both in the shell and core. Taken together, this work provides guidelines for designing LSNAs that exhibit maximal in vivo cancer cell uptake characteristics in the context of a breast cancer model.
Asunto(s)
Ácidos Nucleicos Inmovilizados/metabolismo , Liposomas/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Polietilenglicoles/metabolismo , Animales , Línea Celular Tumoral , Femenino , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/farmacocinética , Liposomas/química , Liposomas/farmacocinética , Ratones Endogámicos BALB C , Peso Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacocinética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/farmacocinética , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Distribución TisularRESUMEN
An interesting phenomenon is described that the fluorescence signal of poly(adenine) (A) DNA-templated gold nanoclusters (AuNCs) is greatly improved in the presence of L-histidine by means of L-histidine-DNA interaction. The modified nanoclusters display strong fluorescence emission with excitation/emission maxima at 290/475 nm. The fluorescence quantum yield (QY) is improved from 1.9 to 6.5%. Fluorescence enhancement is mainly ascribed to the L-histidine-DNA interaction leading to conformational changes of the poly(A) DNA template, which offer a better microenvironment to protect AuNCs. The assay enables L-histidine to be determined with good sensitivity and a linear response that covers the 1 to 50 nM L-histidine concentration range with a 0.3 nM limit of detection. The proposed method has been applied to the determination of imidazole-containing drugs in pharmaceutical samples. A turn-on fluorescent method has been designed for the sensitive detection of L-histidine as well as imidazole-containing drugs on the basis of the L-histidine-DNA interaction.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Histidina/análisis , Nanopartículas del Metal/química , Poli A/química , ADN/metabolismo , Fluorescencia , Oro/química , Histidina/química , Histidina/metabolismo , Imidazoles/análisis , Imidazoles/química , Imidazoles/metabolismo , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Límite de Detección , Poli A/metabolismo , Espectrometría de FluorescenciaRESUMEN
The cell membrane is not only a physical barrier, but also a functional organelle that regulates the communication between a cell and its environment. The ability to functionalize the cell membrane with synthetic molecules or nanostructures would advance cellular functions beyond what evolution has provided. The aim of this Minireview is to introduce recent progress in using synthetic DNA and DNA-based nanostructures for cell-surface engineering. We first introduce chemical conjugation and physical binding methods for monovalent and polyvalent surface engineering. We then introduce the application of these methods for either the promotion or inhibition of cell-environment communication in numerous applications, including the promotion of cell-cell recognition, regulation of intracellular pathways, protection of therapeutic cells, and sensing of the intracellular and extracellular microenvironments. Lastly, we summarize current challenges existing in this area and potential solutions to solve these challenges.
Asunto(s)
Ingeniería Celular/métodos , ADN/metabolismo , Comunicación Celular/fisiología , Membrana Celular/química , Membrana Celular/metabolismo , ADN/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Nanoestructuras/química , Transducción de Señal/fisiologíaRESUMEN
DNA target search is a key step in cellular transactions that access genomic information. How DNA binding proteins combine 3D diffusion, sliding and hopping into an overall search strategy remains poorly understood. Here we report the use of a single molecule DNA tethering method to characterize the target search kinetics of the type II restriction endonuclease NdeI. The measured search rate depends strongly on DNA length as well as salt concentration. Using roadblocks, we show that there are significant changes in the DNA sliding length over the salt concentrations in our study. To explain our results, we propose a model including cycles of 3D and 1D search in which salt concentration modulates the strategy by varying the length of DNA probed per 1D scan. At low salt NdeI makes a single non-specific encounter with DNA followed by an effective and complete 1D scan. At higher salt, NdeI must execute multiple cycles of target search due to the reduced efficacy of 1D search.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Cloruro de Sodio/metabolismo , ADN/química , División del ADN , Difusión , Diseño de Equipo , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Cinética , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
In DNA data storage, the massive sequence complexity creates challenges in repeatable and efficient information readout. Here, our study clearly demonstrated that PCR created significant DNA amplification biases due to its inherent mechanism of inefficient priming, product-as-template, and error-spreading prone, which greatly hinder subsequent applications such as data retrieval in DNA-based storage. To mitigate the amplification bias, we recruited an isothermal DNA amplification by combining strand displacement amplification (SDA) with magnetic beads (MB) DNA immobilization for robust, repeated, and low-bias amplification of DNA oligo pool, comprising over 100 thousand oligos, in a primer-free and low-error-spreading fashion. Furthermore, we introduced oligo pool normalization (OPN), a cost-effective and scalable method for normalizing an oligo pool, by which oligo pools comprising from 256 to 1024 distinct oligos were simply modified with improved Gini-index. Therefore, we believe that the combination of SDA and OPN can provide an ideal amplification mechanism for a low-bias copy of a large oligo pool, which is of vital importance for successful data retrieval in DNA information storage.
Asunto(s)
Oligonucleótidos/metabolismo , Secuencia de Bases , Cartilla de ADN/metabolismo , Biblioteca de Genes , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Magnetismo , Técnicas de Amplificación de Ácido Nucleico , Oligonucleótidos/genéticaRESUMEN
A novel electrochemical nanobiosensor for the detection of miR-155 (as breast cancer biomarker) is introduced . Fe3O4NPs@Ag core-shell nanoparticles were synthesized and their shape and characteristics were confirmed by scanning electron microscope (SEM) imaging, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) methods. Synthesized nanoparticles were applied onto the magnetic bar carbon paste electrode and then the amine-modified anti-miR-155 (single-stranded probes) was applied on the modified electrode surface and upon hybridization with target miR-155, resveratrol (RSV) was eventually applied as an electrochemical label on the double-strand oligonucleotide. Differential pulse voltammetry (DPV) of the oxidation peak of RSV was assumed as the final signal by sweeping potential from 0 to 0.6 V (vs. Ag/AgCl). The fabrication process was optimized through a series of experiments and the optimized process was confirmed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The linear range of the fabricated nanobiosensor was 0.5 fM to 1.0 nM and the detection limit was 0.15 fM. The nanobiosensor was able to pass reproducibility and specificity tests using different types of mismatched target sequences.Spiked real samples of human serum were used to confirm that the nanobiosensor enables detection of miR-155 without any significant interferences from other moieties and molecules. Finally, the molecular dynamics simulation of the RSV interaction with single- and double-stranded oligonucleotide was performed and confirmed the preferential binding of RSV to double-stranded DNA; therefore, it can be used as the electrochemical label of DNA and/or miRNA hybridization-based biosensors. Graphical abstract.
Asunto(s)
Técnicas Biosensibles/métodos , ADN de Cadena Simple/química , Nanopartículas de Magnetita/química , MicroARNs/sangre , Oligodesoxirribonucleótidos/química , Resveratrol/química , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Técnicas Electroquímicas/métodos , Humanos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/genética , Ácidos Nucleicos Inmovilizados/metabolismo , Límite de Detección , MicroARNs/genética , Simulación del Acoplamiento Molecular , Nanocompuestos/química , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Reproducibilidad de los Resultados , Resveratrol/metabolismo , Plata/químicaRESUMEN
Nucleic acid aptamers have been widely used as recognition elements on various biosensing interfaces, but quantitative kinetic/thermodynamic analysis for revealing the aptamer-ligand binding mechanism, which occurs on a liquid-solid interface, has not been realized due to a lack of usable biophysical tools. Herein we apply a resonant microcantilever sensor to continuously record the frequency shift according to the binding-induced mass change on the liquid-solid interface. The frequency-shift curve is used for tracing the reaction process and is fitted with classic equations to calculate a set of kinetic/thermodynamic parameters, such as rate constants (ka = 902.95 M-1 s-1, kd = 0.000141 s-1), equilibrium constants (KD = 1.55 µM), the Gibbs free energy (ΔG° = -32.57 kJ/mol), and the activation energy (Ea = 38.03 kJ/mol) for the immobilized aptamer and free ATP. This quantitative analysis method is label-free, calibration-free, and highly sensitive. The kinetic/thermodynamic parameter detection method provides new resolution to the in-depth understanding of the ligand-aptamer interaction on the liquid-solid interface for biosensing or lab-on-a-chip applications.
Asunto(s)
Aptámeros de Nucleótidos/química , Ácidos Nucleicos Inmovilizados/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Aptámeros de Nucleótidos/metabolismo , Técnicas de Química Analítica/métodos , Ácidos Nucleicos Inmovilizados/metabolismo , Indoles/química , Cinética , Ligandos , Microesferas , Polímeros/química , TermodinámicaRESUMEN
Norovirus is one of the leading causes of gastroenteritis, acute vomiting, intense diarrhoea, acute pain in the stomach, high fever, headaches, and body pain. Conventional methods of detection gave us very promising results but had disadvantages such as low sensitivity, cost ineffectiveness, reduced specificity and selectivity, etc. Therefore, biosensors can be a viable alternative device which can overcome all setbacks associated with the conventional method. An electrochemical sensor based on oxidized graphitic carbon nitride (Ox-g-C3N4) modified electrochemical paper-based analytical device (ePAD) was fabricated for the detection of norovirus DNA. The synthesized Ox-g-C3N4 nanosheets were characterized by field emission scanning electron microscopy (FESEM), X-ray Diffraction (XRD), UV-Vis spectroscopy and X-Ray Photoelectron Spectroscopy. The capture probe DNA (PDNA) modified electrodes were characterized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). These two characterization techniques were also employed to find the optimal scan rate, response time and temperature of the fabricated sensor. The fabricated biosensor showed a limit of detection (LOD) of 100 fM. Furthermore, the specificity of the reported biosensor was affirmed by testing the response of capture probe DNA with oxidized graphitic carbon nitride (PDNA/Ox-g-C3N4) modified ePAD on the introduction of a non-complimentary DNA. The fabricated ePAD sensor is easy to fabricate, cost effective and specific, and requires a minimum analysis time of 5 s.
Asunto(s)
Técnicas Biosensibles/métodos , Grafito/química , Compuestos de Nitrógeno/química , Norovirus/genética , Papel , ARN Viral/análisis , Técnicas Biosensibles/instrumentación , Sondas de ADN/química , Sondas de ADN/metabolismo , Técnicas Electroquímicas/métodos , Electrodos , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Límite de Detección , Azul de Metileno/química , Nanoestructuras/química , Hibridación de Ácido Nucleico , ARN Viral/metabolismoRESUMEN
A Y-type-DNA-functionalized nanogold probe was synthesized to identify telomerase and trigger drug release in cancer cells. This system involved a DNA-functionalized nanogold probe centered on gold nanoparticles with a dense modification of Y-type DNA molecular beacons on the surface. The Y-type DNA molecular beacons consisted of telomerase primers (TPs), a FAM-labeled single-strand DNA (Mismatch-DNA), and a single-strand DNA of two templates (Linker-DNA1 and Linker-DNA2). Doxorubicin (Dox), an anti-cancer drug molecule, was inserted into DNA double strands. When telomerase existed, TP was extended and the Mismatch-DNA was released, leading to the emission of fluorescent light from FAM while releasing Dox. This probe specifically detected cancer cells and did not affect normal cells. This drug delivery system will reduce the tumour size and cause minimal injury to normal tissues.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Oro/metabolismo , Ácidos Nucleicos Inmovilizados/metabolismo , Telomerasa/metabolismo , Animales , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapéutico , Portadores de Fármacos/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones Desnudos , Nanopartículas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Apolipoprotein E4 (ApoE4) has a key role on the onset and progression of Alzheimer's disease (AD), since it favours the deposition of toxic amyloid-beta (Aß) aggregates in the brain. These effects might result from the interaction between ApoE4 and specific DNA promoters related to cellular autophagy pathways and to the expression of neuroprotective proteins, like sirtuin-1. Herein, we modified gold electrodes with mixed self-assembled monolayers of 6-mercapto-1-hexanol and thiolated DNA oligonucleotides related to CLEAR (associated with autophagic processes that enable the clearance of toxic species, such as Aß) and SirT1 (related to the expression of sirtuin-1) promoter sequences. The interactions of the immobilized DNA sequences with isoforms of ApoE (ApoE4/ApoE3/ApoE2) were investigated by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) measurements. By monitoring current and charge transfer resistance (Rct) variations, CLEAR showed to interact specifically with ApoE4, whereas SirT1 showed a higher affinity to ApoE4 compared to ApoE3 and ApoE2. To the best of our knowledge, this is the first report about the application of electrochemical techniques to investigate the sequence-specific interaction between ApoE isoforms and CLEAR and SirT1 oligonucleotides.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apolipoproteínas E/metabolismo , Sirtuina 1/genética , Enfermedad de Alzheimer/genética , Apolipoproteína E2/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Secuencia de Bases , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Electrodos , Humanos , Ácidos Nucleicos Inmovilizados/genética , Ácidos Nucleicos Inmovilizados/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismoRESUMEN
Vascular endothelial cells are essential to vascular function and maintenance. Dysfunction of these cells can lead to the development of cardiovascular disease or contribute to tumorigenesis. As such, the therapeutic modulation and monitoring of vascular endothelial cells are of significant clinical interest, and several endothelial-specific ligands have been developed for drug delivery and the monitoring of endothelial function. However, the application of these ligands has been limited by their high cost and tendency to induce immune responses, highlighting a need for alternate methods of targeting vascular endothelial cells. In the present study, we explore the therapeutic potential of DNA aptamers. Using cell-SELEX technology, we identified two aptamers with specific binding affinity for vascular endothelial cells and propose that these molecules show potential for use as new ligands for drug and biomarker research concerning vascular endothelial cells.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Células Endoteliales/metabolismo , Ácidos Nucleicos Inmovilizados/metabolismo , Animales , Biomarcadores/metabolismo , Carcinogénesis/metabolismo , Enfermedades Cardiovasculares/metabolismo , Femenino , Ligandos , RatasRESUMEN
Here, we demonstrate use of a Mg2+-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.
Asunto(s)
ADN Catalítico/metabolismo , Ácidos Nucleicos/metabolismo , Resinas Acrílicas/química , Resinas Acrílicas/metabolismo , Geles/química , Geles/metabolismo , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Magnesio/química , Magnesio/metabolismo , Técnicas de Amplificación de Ácido Nucleico , Ácidos Nucleicos/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Biosensibles/métodos , ADN-Topoisomerasas de Tipo I/análisis , Ácidos Nucleicos Inmovilizados/metabolismo , Mycobacterium/aislamiento & purificación , Patología Molecular/métodos , Proteínas Bacterianas/metabolismo , Biomarcadores/análisis , Biomarcadores/metabolismo , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/metabolismo , Colorantes Fluorescentes/química , Humanos , Ácidos Nucleicos Inmovilizados/química , Mycobacterium/enzimología , Sensibilidad y EspecificidadRESUMEN
We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.
Asunto(s)
5-Metilcitosina/análisis , Biotina/química , Ensayo de Inmunoadsorción Enzimática , Ácidos Nucleicos Inmovilizados/química , Mecloretamina/química , 5-Metilcitosina/inmunología , Animales , Biotina/metabolismo , Encéfalo/metabolismo , Metilación de ADN , Epigénesis Genética , Genoma , Ácidos Nucleicos Inmovilizados/metabolismo , Intestino Delgado/metabolismo , Mecloretamina/metabolismo , Ratones , Análisis de Secuencia de ADN , Estreptavidina/química , Estreptavidina/metabolismoRESUMEN
A novel and highly sensitive method based on bio-barcode with rolling circle amplification (RCA) was developed for the detection of T-2 toxin. Gold nanoparticles (AuNPs) were modified with anti-T-2 monoclonal antibody and single-stranded thiol-oligonucleotides (SH-ssDNAs) and magnetic microparticles (MMPs) coated with T-2 antigen. The T-2 toxin competes with the antigen on MMPs for the anti-T-2 antibody on AuNPs. Then, the isolating complex system was separated by a magnetic field, and the DNA of the probes was released after washing in dithiothreitol solution. The barcode DNA via RCA and products were stained by SYBR Green I and then detected by fluorescence spectrophotometry. The optimized method was performed on oats, millet, flour, and other substances. This method exhibits a low limit of detection (0.26â¯pgâ¯mL-1) and linear range of 0.002-200â¯ngâ¯mL-1. Moreover, the approach offers good recovery and relative standard deviations ranging from 88.65% to 10.04% and 0.6%-13.1%, respectively. In conclusion, this method exhibits potential for use as an ultrasensitive assay for the detection of a variety of small molecules in complex matrices.
Asunto(s)
ADN de Cadena Simple/química , Contaminación de Alimentos/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , Espectrometría de Fluorescencia , Toxina T-2/análisis , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Benzotiazoles , ADN de Cadena Simple/metabolismo , Diaminas , Oro/química , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Límite de Detección , Magnetismo , Nanopartículas del Metal/química , Compuestos Orgánicos/química , Quinolinas , Toxina T-2/inmunologíaRESUMEN
Detecting and monitoring the pathogens with high selectivity and sensitivity is critical for public health. In the present study, we demonstrated a specific analytical strategy for sensitive detection of Leishmania infantum genome. The developed sensor utilized toluidine blue as a hybridization indicator and a Leishmania infantum-specific capture DNA sequence immobilized on a high-surface area gold nanostructure as an electrochemical transducer. The produced analytical response was based on the hybridization of the single-stranded DNA from the target with the immobilized DNA sequence at the electrode surface. The developed DNA sensor in this study was successfully employed to detect a synthetic Leishmania infantum target sequence in a wide concentration range from 1â¯×â¯10-18 to 1â¯×â¯10-10â¯molâ¯L-1 with a detection limit of 0.2â¯amolâ¯L-1 with the ability to discriminate the target sequence from mismatched sequences. Moreover, the designed DNA sensor showed a good reproducibility and stability during repeated regeneration and hybridization cycles. The DNA sensor could detect Leishmania infantum genome in a wide concentration range from 15 to 50â¯ng⯵L-1 with a detection limit of 29â¯ng⯵L-1. Furthermore, clinical trials confirmed the applicability of the developed DNA sensor for practical applications.
Asunto(s)
ADN Bacteriano/análisis , Técnicas Electroquímicas , Oro/química , Leishmania infantum/aislamiento & purificación , Nanopartículas del Metal/química , ADN Bacteriano/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Electrodos , Genoma Bacteriano , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Leishmania infantum/genética , Límite de Detección , Hibridación de Ácido Nucleico , Reproducibilidad de los ResultadosRESUMEN
Fluorescence enhancement by plasmonic nanostructures enables the optical detection of single molecules with weak fluorescence, extending the scope of molecular fluorescence imaging to new materials and systems. In this work, we study single-molecule fluorescence enhancement by individual gold nanorods exploiting a DNA-based transient binding technique. Single molecules are attached to short DNA oligomers that can reversibly hybridize to their complementary docking DNA strands immobilized on the surface of gold nanorods or the glass substrate next to gold nanorods. This method continuously refreshes the single molecule in the near field of the gold nanorod, and enables a study of fluorescence enhancement at a well-defined position, with long dwell time and without limitation by photobleaching. Docking strands attached to the glass substrate are found to be more photo-stable. We find over 3000-fold fluorescence enhancement of single molecules of IRDye800CW, a near-infrared dye with a low quantum yield of 7%. This strong enhancement, consistent with numerical simulations, arises from the combined effect of local field enhancement and the competition between radiative and nonradiative decay rate enhancements.
Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Oro/química , Nanotubos/química , Sitios de Unión , ADN/metabolismo , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Microscopía Confocal , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Espectroscopía Infrarroja CortaRESUMEN
The complete formation of stem-loop (i.e., hairpin) configuration on chip surface is of particular importance for the application of hairpin DNA (hpDNA) in building biosensors for various analytes with optimized performance. We report herein a convenient electrochemical protocol for evaluating the yield of hairpin DNA conformations upon self-assembly on electrode surface. As of the different hydrolysis capability of Exonuclease I (Exo I) toward single-stranded DNA (ssDNA) and hpDNA, we can selectively remove ssDNA from electrode but retain hpDNA strands; based on the changes in the cyclic voltammetric (CV) responses using [Ru(NH3)6]3+ as redox indicators, we can then determine the fraction of hairpin configurations in mixed DNA self-assembled monolayers (SAMs). It was discovered that the molar fraction of hairpin configuration formed on the surface is considerably lower than that in the binary deposition solution (containing both ssDNA and hpDNA). The accuracy of the Exo I-assisted electrochemical quantitative protocol has been validated by standard DNA hybridization experiments; the relationship between the overall DNA packing density and the yield of hairpin configurations was also evaluated. More importantly, taking HIV-1 gene detection as a trial system, the hpDNA-based biosensor shows significantly improved detection limit and broadened response range upon the background reduction by Exo I-catalyzed hydrolysis.
Asunto(s)
Técnicas Biosensibles/métodos , ADN Viral/química , Exodesoxirribonucleasas/metabolismo , VIH-1/genética , Ácidos Nucleicos Inmovilizados/química , Ácidos Nucleicos Inmovilizados/metabolismo , Secuencias Invertidas Repetidas , Secuencia de Bases , ADN Viral/genética , ADN Viral/metabolismo , Electroquímica , Hidrólisis , Ácidos Nucleicos Inmovilizados/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Propiedades de SuperficieRESUMEN
The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.