DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers). 相似文献
DNA microarray enables the analysis of DNA or mRNA expression levels, but it has not been possible to completely understand life using obtained information. Consequently, protein or peptide arrays have attracted much interest. Since the development of a practical protein microarray is still far away in light of handling difficulties, the peptide microarray is a promising tool for analyzing protein functions. We have developed a peptide microarray to detect protein kinase activity in cell lysate. All substrate peptides for kinases were immobilized chemoselectively on amino-coated glass slides. After phosphorylation of the immobilized peptides, phosphorylation was detected by fluorescence imaging. We detected the protein kinase activities, including that in cell lysate, in response to drug stimulation. Therefore, this peptide microarray would be useful for a high-throughput kinase assay of intracellular signals and would be applicable to drug screening. 相似文献
The use of aptamers in various analytical applications as molecular recognition elements and alternative to antibodies has led to the development of various platforms that facilitate the sensitive and specific detection of targets ranging from small molecules and proteins to whole cells. The goal of this work was to design a universal and adaptable sandwich-type aptasensor exploiting the unique properties of DNA binding proteins. Specifically, two different enzyme-DNA binding protein conjugates, GOx-dHP and HRP-scCro, were used for the direct detection of a protein using two aptamers for target capture and detection. The specific dsDNA binding sequence for each DNA binding protein tag was incorporated in the form of a hairpin at one end of each aptamer sequence during the synthesis step. Detection was accomplished by an enzymatic (GOx/HRP) cascade reaction after the binding of each enzyme conjugate to its corresponding binding sequence on each aptamer. The proposed sandwich-type aptasensor was validated for the detection of thrombin, which is one of the most commonly used model targets with known dual aptamers. The limit of detection accomplished was 0.92 nM which is comparable with other colorimetric platforms reported in the literature. The sensitivity of the aptasensor was easily modulated by changing the number of dsDNA binding sites incorporated in the aptamer sequences, thus controlling the enzyme stoichiometry. Finally, the potential use of the proposed sensing approach for real sample testing was demonstrated using spiked human plasma and no significant matrix effects were observed when up to 2% plasma was used.
We applied Systematic Evolution of Ligands by EXponential enrichment using Small Cell Lung Cancer (SCLC) cells. A DNA aptamer was identified and evaluated by fluorescent confocal microscopy and flow cytometry. Our results showed that the DNA aptamer binds to molecules that exist predominantly on target SCLC cell surfaces compared with other types of SCLC cells. 相似文献
Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from complex libraries of synthetic nucleic acids by an iterative process of adsorption, recovery and amplification. This review described the in vitro process to obtain aptamers (SELEX). It mentions the main characteristics of these molecules (i.e. affinity, specificity and stability). Moreover, it discusses advantages over antibodies. It reports potential applications of aptamers in analytical and diagnostic assays as biocomponents of biosensors (aptasensors) and allosteric ribozymes (aptazymes). 相似文献
High‐affinity aptamers for important signal transduction proteins, i.e. Cdc42‐GTP, p21‐activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase‐related Cdc42‐binding kinase) α were successfully selected in the low micro‐ to nanomolar range using non‐systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naïve DNA library. In the non‐SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non‐equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non‐SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (Kd) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non‐SELEX for the selection of numerous unique sequences with high selectivity. 相似文献
Aptamer is an oligonucleotide chain with specific binding ability to protein and other targets,which is widely used in ma ny fields.Because of its ability to screen the premise of unknown targets,it can be used to discover some novel tumor markers,i.e.,membrane proteins that are specifically highly expressed on the surface of tumor cells.Tumor markers can be used in many fields such as early diagnosis and treatment,and a new type of tumor marker proved to be effective can significantly improve the therapeutic effect of such tumors.However,further characterization of newly acquired membrane proteins is essential for their clinical use as tumor markers.This review first briefly introduced the process of obtaining novel tumor markers from nucleic acid aptamers.Next,the commonly used protein characterization methods could be used as a technical means to identify membrane protein targets corresponding to tumor cell aptamers,to clarify the principles,advantages and disadvantages of various means,and to analyze the most suitable situations for various experimental methods.Finally,the outlook was made and the characterization methods that should be used in such experiments were summarized. 相似文献
In this research, we used the non-SELEX method to successfully select an aptamer that binds to the protein target (bovine catalase) with a K(D) value in the low micro molar range. The time window was determined for the target and aptamer library by optimizing the buffer conditions using 3 × Tris-glycine-potassium phosphate (TGK) buffer as the run buffer and 1× TGK as the selection buffer to give the biggest complex peak. Fractions were collected by multistep nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)-based partitioning for three rounds of selection. The fractions from each round were enriched using PCR and the progress of selection was monitored using bulk affinity analysis. Fraction 2 was determined to have the optimal bulk affinity (0.75 μM) and this enriched library was cloned and sequenced giving four aptamer sequences. These sequences were verified using affinity capillary electrophoresis (CAT 1 0.237 μM) and fluorescence intensity measurements (CAT 1 0.430 μM). The specificity of the aptamer was also determined by fluorescence intensity measurements. The results showed that the aptamer with the highest binding affinity showed at least a 100-fold decrease in affinity toward four other proteins (i.e. lysozyme, trypsinogen, chymotrypsinogen A, and myoglobin) tested and this confirmed that the aptamer exhibited a distinct specificity toward bovine catalase. This aptamer will be useful in biosensing, Western blot, and biomarker identification. 相似文献
Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by Fusarium graminearum on maize and barley. Because most current methods of ZEN detection rely on the use of low-stability antibodies or expensive equipment, we sought to develop a rapid, low-cost determination method using aptamers instead of antibodies as the specific recognition ligands. This work describes the isolation and identification of single-stranded DNA (ssDNA) aptamers recognizing ZEN using the modified systematic evolution of ligands by exponential enrichment methodology based on magnetic beads. After 14 rounds of repeated selection, a highly enriched ssDNA library was sequenced and 12 representative sequences were assayed for their affinity and specificity. The best aptamer, 8Z31, with a dissociation constant (Kd) of 41?±?5 nM, was successfully applied in the specific detection of ZEN in binding buffer and in real samples based on a magnetic separation/preconcentration procedure. This analytical method provided a linear range from 3.14?×?10?9 to 3.14?×?10?5 M for ZEN, and the detection limit was 7.85?×?10?10 M. The selected aptamers are expected to be used in the potential development of affinity columns, biosensors, or other analytical systems for the determination of ZEN in food and agricultural products.
Figure
Determination of dissociation constant (Kd) and specificity of aptamers recognizing zearalenone 相似文献
Anabolic androgenic steroids (AAS) are frequently abused in human and animal sports as performance-enhancing drugs, and consequently their use is controlled by international sports authorities. Testosterone is one of the most frequently used AAS, and therefore the accurate determination of its levels in biological fluids is very important. The authors describe the selection of testosterone-binding aptamers performed using a classic SELEX approach with the target immobilized on magnetic beads. Counter selections with structurally similar steroids were implemented at different stages. Pools from different selection rounds were sequenced with Next Generation Sequencing and ten aptamer candidates were selected for further characterization. Low nanomolar range dissociation constants were calculated by a bead-based PCR assay and verified by microscale thermophoresis. Future work will focus on the development of aptamer-based platforms for the sensitive detection of testosterone in biological samples and the validation of these assays for the rapid screening of suspicious samples.
Non‐systematic evolution of ligands by exponential enrichment and other capillary‐based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary‐based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two‐step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary‐based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a KD of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two‐step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments. 相似文献
Identifying cells associated with specific disease states is critically important for the early detection and diagnosis of cancer. To facilitate this task, molecular probes, which bind biomarkers that are either specifically or differentially expressed in diseased cells relative to healthy cells, provide a simple and effective method. This review focuses on the use of DNA aptamers as molecular probes for cancer cells. These aptamers are created by means of the cell-based Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, which uses whole disease cells as targets. We describe at length the steps of the cell-SELEX process and discuss several applications for the aptamers, including profiling leukemia patient samples and discovering cell-surface cancer biomarkers. We conclude with a discussion of an aptamer-conjugated nanoparticle enrichment and detection scheme. 相似文献
Single-drop analysis of two different real sample solutions (2 μL) while simultaneously monitoring the activity of two sets
of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip
(CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane
(PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the
fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5–8.2
(% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP
with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases
in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously
carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested
the usefulness of the CAs-CHIP in the field of drug discovery.
Figure Single drop analysis of two real sample solutions including various different proteases using a single microfluidic device
was achieved 相似文献
Researchers frequently use two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D‐PAGE method, which ‘inverts’ the dimension of protein separation of the conventional 2D‐PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first‐dimension separation was conducted on a gel‐free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second‐dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D‐PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D‐PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D‐PAGE. Overall, the i2D‐PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research. 相似文献
A highly sensitive surface-enhanced Raman (SERS)-based method for detection of influenza viral nucleoproteins is described. The intrinsic SERS spectrum of the aptamer-nucleoprotein complex provides direct evidence of binding between a polyvalent anti-influenza aptamer and the nucleoproteins of three influenza strains. 相似文献