Selection,Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.     

Assays for cytokines using aptamers

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.     

Potent inhibition of human influenza H5N1 virus by oligonucleotides derived by SELEX

New therapeutics are urgently needed for the treatment of pandemic influenza caused by H5N1 influenza virus mutants. Aptamer was a promising candidate for treatment and prophylaxis of influenza virus infections. In this study, systemic evolution of ligands through exponential enrichment (SELEX) was used to screen DNA aptamers targeted to recombinant HA1 proteins of the H5N1 influenza virus. After 11 rounds of selection, DNA aptamers that bind to the HA1 protein were isolated and shown to have different binding capacities. Among them, aptamer 10 had the strongest binding to the HA1 protein, and had an inhibitory effect on H5N1 influenza virus, as shown by the hemagglutinin and MTT assays. These results should aid the development of new drugs for the prevention and control of influenza virus infections.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

A novel lipopolysaccharide-antagonizing aptamer protects mice against endotoxemia

A growing number of researchers have recognized the importance of using lipopolysaccharide (LPS) as target for the prevention and treatment of sepsis. However, no drugs targeting LPS have been applied clinically. In this study, LPS-inhibiting aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and their therapeutic effects for experimental sepsis were observed. After 12 rounds of screening, 46 sequences were obtained. Primary structure analysis indicated that they had identical sequences, partly conserved sequences, or non-conserved sequences. Secondary structure analysis showed these sequences usually contained hairpin or stem-loop structures. Aptamer 19 significantly decreased NF-κB activation of monocytes challenged by LPS and reduced the IL-1 and TNF-α concentration in the media of LPS-challenged monocytes. Furthermore, aptamer 19 significantly increased the survival rate of mice with endotoxemia. The results suggest that a novel LPS antagonizing aptamer was obtained by SELEX, which successfully treated experimental sepsis.     

Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting

Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (K _d value) of 1.73?±?0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 10²–10³?CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.     

In vitro selection of a DNA aptamer targeted against Shigella dysenteriae

To identify DNA aptamers demonstrating binding specificity for Shigella dysenteriae, a whole-bacterium Systemic Evolution of Ligands by Exponential enrichment (SELEX) method was applied to a combinatorial library of single-stranded DNA (ssDNA) molecules. After several rounds of selection using S. dysenteriae as the target, the highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homologies and similarities in the secondary structures. Aptamer S 1, which showed particularly high binding affinity in preliminary studies, was chosen for further characterisation. This aptamer displayed a dissociation constant (K_d value) of 23.47 ± 2.48 nM. Binding assays to assess the specificity of aptamer S 1 showed high binding affinity for S. dysenteriae and low apparent binding affinity for other bacteria. The ssDNA aptamers generated may serve as a new type of molecular probe for microbial pathogens, as it has the potential to overcome the tedious isolation and purification requirements for complex targets.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

应用核酸适配子检测细胞因子的新方法—ELONA法

以人肿瘤坏死因子(Human tumor necrosis factor,hTNF—α)特异性的核酸适配子为检测分子建立了酶联寡聚核苷酸吸附试验(Enzyme—linked Oligonucleotide assay,ELONA)方法,用于hTNF—α的检测。通过SELEX(Systematic Evolution of Ligands by Exponential Enrichment)方法从随机RNA库中筛选到与hTNF—α特异结合的RNA适配子。根据其序列,用体外转录方法合成生物素标记的RNA适配子,并对其进行了氨基修饰以增加其稳定性。以hTNF—α的单克隆抗体为捕获分子,生物素标记的hTNF—α特异性RNA适配子为检测分子建立了ELONA方法,并对这种检测方法的灵敏度、精密度和准确度等进行了分析。同时用ELONA和ELISA方法检测了正常人血清中的hTNF—α水平,并对检测结果进行比较。结果显示,ELONA方法的灵敏度为100pg／mL,具有较好的精密度和准确度。ELONA法的检测结果与ELISA法检测结果基本一致。该方法适用于血清、细胞培养上清等多种生物标本中各种细胞因子及其它蛋白的检测。     

Advancing Aptamers as Molecular Probes for Cancer Theranostic Applications—The Role of Molecular Dynamics Simulation

Theranostics cover emerging technologies for cell biomarking for disease diagnosis and targeted introduction of drug ingredients to specific malignant sites. Theranostics development has become a significant biomedical research endeavor for effective diagnosis and treatment of diseases, especially cancer. An efficient biomarking and targeted delivery strategy for theranostic applications requires effective molecular coupling of binding ligands with high affinities to specific receptors on the cancer cell surface. Bioaffinity offers a unique mechanism to bind specific target and receptor molecules from a range of non‐targets. The binding efficacy depends on the specificity of the affinity ligand toward the target molecule even at low concentrations. Aptamers are fragments of genetic materials, peptides, or oligonucleotides which possess enhanced specificity in targeting desired cell surface receptor molecules. Aptamer–target binding results from several inter‐molecular interactions including hydrogen bond formation, aromatic stacking of flat moieties, hydrophobic interaction, electrostatic, and van der Waals interactions. Advancements in Systematic Evolution of Ligands by Exponential Enrichment (SELEX) assay has created the opportunity to artificially generate aptamers that specifically bind to desired cancer and tumor surface receptors with high affinities. This article discusses the potential application of molecular dynamics (MD) simulation to advance aptamer‐mediated receptor targeting in targeted cancer therapy. MD simulation offers real‐time analysis of the molecular drivers of the aptamer‐receptor binding and generate optimal receptor binding conditions for theranostic applications. The article also provides an overview of different cancer types with focus on receptor biomarking and targeted treatment approaches, conventional molecular probes, and aptamers that have been explored for cancer cells targeting.     

综述与专论: 核酸适配体筛选与亲和力评价技术主要研究方向、进展与挑战

核酸适配体是一类具有特异性分子识别能力的单链DNA或者RNA分子,通过指数富集的配体系统进化技术（SELEX）筛选得到。核酸适配体相比抗体具有热稳定性高、便于化学合成与修饰、免疫原性低等优点,在生物分析、生物医学、生物技术等众多领域引起广泛关注。高质量的核酸适配体是应用的基础,然而目前能够满足实际应用的核酸适配体数量还非常有限。如何获得高亲和力、高特异性、高体内稳定性的核酸适配体是核酸适配体领域的技术瓶颈。本文首先简单介绍了SELEX技术的基本原理和核酸库的设计、筛选过程监控、次级文库制备、测序和候选适配体筛选等关键步骤。接着归纳总结了30多年来核酸适配体筛选技术的6个主要研究方向、研究进展和局限性。这6个主要研究方向分别是提高适配体特异性的筛选方法、提高适配体稳定性（抗核酸酶降解能力）的筛选方法、快速筛选方法、复杂靶标适配体筛选方法、小分子靶标适配体筛选方法、提高适配体亲和力的筛选方法。其中快速筛选技术是长期以来持续关注的研究方向,几乎所有物理分离手段都已用于提高SELEX的筛选效率。最近,高效化学反应与SELEX技术的结合为核酸适配体的快速筛选提供了新的策略。本文随后对适合小分子靶标核酸适配体筛选的3类方法进展和存在的问题进行了重点评述。这3类方法分别是基于靶标固定的筛选技术、基于文库固定的筛选技术（捕获-SELEX,Capture-SELEX）和均相筛选技术（氧化石墨烯-SELEX,GO-SELEX）。基于靶标固定的筛选技术尽管存在空间位阻等众多问题,由于其操作的简单性,目前依然应用广泛。近年来Capture-SELEX应用广泛。结合36种靶标适配体的筛选实验条件（文库设计、正筛靶标浓度、负筛靶标的选择和浓度）和所获得的适配体的亲和力（K_D,解离常数,dissociation constant）和特异性,对Capture-SELEX的实验条件与适配体性能的关系进行了讨论。统计数据表明,降低正筛靶标浓度有利于提高适配体的亲和力,但不是必要条件。负筛选是目前提高适配体特异性的主要技术手段,但适配体的特异性还不能满足实际需求。负筛选靶标及其浓度的选择差异很大,而且36种靶标中有20种靶标的适配体筛选没有进行负筛选。如何提高核酸适配体的特异性是目前小分子靶标核酸适配体所面临的难题,急需寻找新的策略。本文还列表归纳了近三年利用GO-SELEX进行的13种小分子靶标的实验条件和所获得的适配体的K_D和特异性。统计数据表明,GO-SELEX比Capture-SELEX所需要的筛选轮数少,两种方法所获得的适配体的亲和力多在纳摩尔每升水平。Capture-SELEX相对较低的筛选效率应该主要由于文库的自解离问题。核酸适配体的亲和力评价是候选核酸适配体结构与性能评价的重要组成部分。常用的核酸适配体亲和力评价技术包括基于分离、基于固定、均相体系三大类十多种方法。假阳性和假阴性是各种评价技术都有可能存在的问题。本文以纳米金比色法和等温热滴定技术为例评述技术进展,讨论导致不同亲和力评价技术结果不一致性问题的根本原因。本文最后对核酸适配体筛选技术、亲和力评价技术和技术的标准化的未来发展趋势进行了展望。     

In vitro lectin-mediated selection and characterization of rHuEPO-α-binding ssDNA aptamers

A lectin-mediated affinity chromatographic SELEX technique was developed to generate functional ssDNA aptamers for recombinant human erythropoietin-α (rHuEPO-α), an important pharmaceutical glycoprotein for the first time. Secondary structure analysis of the aptamer clones from sequential 6th, 7th, and 8th rounds showed that a certain fragment 'CGAGAT' in the 3' primer region could be used to trace increasing evolution stringency from its hybridization at different locations, in which a specific hybridization with its complement in the 5' primer region (aptamer 807) was evolved as the prevalent one with the simplest and most common motif. Characteristics of the aptamers with lower Gibbs' free energies and K(d) values (nM) were investigated. For aptamer 813, the minimer 813-42nt formed by the random and primer regions was indispensable for the specific binding with rHuEPO-α. While for aptamer 807, only the random region, that is, 807-39nt, was the functional motif. Further experiments of methylation, site-directed mutation and length variation showed that the loop of aptamer 807-39nt was the key region for binding with rHuEPO-α, and the stem should be considered as a stabilizing part. Lower cross-reactivity of aptamer 807-39nt was observed with human normal urothelium tissues than the anti-EPO monoclonal antibody AE7A5. Aptamer 807-39nt also exhibited a specific recognition for human bladder carcinoma cells and human urothelium tumors, which might provide a novel way to probe such tumors with overexpressed EPOs.     

Selection and characterization of DNA aptamers specific for Listeria species

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37 °C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.     

基于HT-SELEX筛选的节球藻毒素-R适配体的鉴定

节球藻毒素-R（nodularin-R,NOD-R）是具有强烈肝毒性的蓝藻毒素。然而,现有的NOD-R检测技术均存在一定的局限性,亟待开发一种新的检测方法。本文利用指数富集的配基系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术结合高通量测序（HT-SELEX）,筛选出NOD-R特异的高亲和力适配体。生物膜干涉（biolayer interferometry,BLI）技术对适配体的鉴定结果表明,适配体H62与NOD-R之间的亲和力最高,达到了168 nM。而且,该适配体不与其他毒素结合,具有较高的特异性。以上结果表明,适配体H62有望作为NOD-R检测新方法——适配体生物传感器的分子识别元件。     

Characterisation of aptamer–target interactions by branched selection and high-throughput sequencing of SELEX pools

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10¹⁶ different RNA or DNA sequences by 5–10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2′-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.     

Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.     

SELEX Aptamer Used as a Probe to Detect Circulating Tumor Cells in Peripheral Blood of Pancreatic Cancer Patients

Many studies have shown that the quantity and dynamics of circulating tumor cells (CTCs) in peripheral blood of patients afflicted with solid tumours have great relevance in therapeutic efficacy and prognosis. Different methods based on various strategies have been developed to isolate and identify CTCs, but their efficacy needs to be improved because of the rarity and complexity of CTCs. This study was designed to examine the possibility of using a SELEX aptamer (BC-15) as a probe to identify rare CTCs out of background nucleated cells. Aptamer BC-15 was selected from a random oligonucleotide library screened against human breast cancer tissue. Fluorescence staining showed that BC-15 had a high affinity for nuclei of human cancer cell lines of various origins as well as CTCs isolated from pancreatic cancer patients, whereas its binding capacity for non-tumor breast epithelial cells and leukocytes was almost undetectable. BC-15⁺/CD45^- cells in cancer patient blood were also found to be cytokeratins 18-positive and aneuploid by immunofluorescence staining and fluorescent in situ hybridization, respectively. Finally, the aptamer method was compared with the well-established anti-cytokeratin method using 15 pancreatic cancer patient blood samples, and enumeration indicated no difference between these two methods. Our study establishes a novel way to identify CTCs by using a synthetic aptamer probe. This new approach is comparable with the anti-cytokeratin-based CTC identification method.     

化学修饰寡核苷酸在核酸配基扩增技术中的应用

核酸酸基扩增技术（SELEX)可从极大容量的随机寡核苷酸文库中筛选得到与靶分子高特异性和高亲和力结合的核酸配基。对寡核苷酸进行化学修饰,可以提高核酸配基的稳定性,增加其功能多样性及生物利用度。SELEX在基础研究、诊断和治疗试剂的研制及药物筛选等领域有广泛用途。