Chip-based detection of hepatitis C virus using RNA aptamers that specifically bind to HCV core antigen

The development of reagents with high affinity and specificity to the antigens of hepatitis C virus (HCV) is important for the early stage diagnosis of its infection. Aptamers are short, single-stranded oligonucleotides with the ability to specifically recognize target molecules with high affinity. Herein, we report the selection of RNA aptamers that bind to the core antigen of HCV. High affinity aptamers were isolated from a 10(15) random library of 60 mer RNAs using the SELEX procedure. Importantly, the selected aptamers specifically bound to the core antigen, but not to another HCV antigen, NS5, in a protein chip-based assay. Using these aptamers, we developed an aptamer-based biosensor for HCV diagnosis and detected the core antigen from HCV infected patients' sera with good specificity. This novel aptamer-based antigen detection sensor could be applied to the early diagnosis of HCV infection.     

综述与专论: 基于核酸适配体传感器的即时检测（POCT）技术

即时检测（point-of-care testing，POCT）是一种检测成本低、检测速度快、准确度高、能自我采样获得临床诊断结果的新型诊断技术。该技术在临床诊断、病情监控与疫情防控等领域发挥了重要作用。核酸适配体是一种能够特异性识别多种靶标的分子探针，具有易合成、批间差异小、易实现信号放大等突出优势，是生物医学传感器中重要的分子识别元件。本文概述了核酸适配体探针的现有筛选方法和进展，总结了核酸适配体POCT传感器信号放大策略，着重介绍了各类核酸适配体传感器在POCT领域的应用现状，并对核酸适配体POCT传感器的发展前景进行了展望。     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

Expanding the application potential of DNA aptamers by their functionalization

Side-by-side development of two competing technologies for obtaining affinity antibody-based and aptamer-based molecules opens new horizons for the creation of diagnostic and therapeutic agents of extremely high efficiency. Benefits of aptamers, such as relatively small size and selection simplicity, have been jeopardized for a long time by their intrinsic downsides, i.e., obscure process of obtaining aptamers against certain targets because of a low diversity of functional groups (purine and pyrimidine bases) in DNA and RNA aptamers. Another side effect of the aptamer technique inherent to the traditional SELEX method is unspecific enrichment with aptamers with high affinity to off-target reaction components. Today, due to current progress in the development of new technology methods and chemical coupling reactions, the modern aptamer technology helps to avoid its disadvantages and become capable of being the source of new diagnostic and therapeutic tools, which are properly unique in their efficiency. The review focuses on modern methods of increasing efficiency of the aptamer selection and on synthetic nucleotide modifications, which make it possible to prepare high-affinity aptamers against traditionally ‘hard’ targets.     

Therapeutic aptamers: developmental potential as anticancer drugs

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]     

Electrochemical aptamer-based sensors

The valuable properties of aptamers, such as specificity, sensitivity, stability, cost-effectiveness and design flexibility, have favoured their use as biorecognition elements in biosensor development. These synthetic affinity probes can be developed for almost any target molecule, covering a wide range of applications in fields such as clinical diagnosis and therapy, environmental monitoring and food control. The combination of aptamers with high-performance electrochemical transducers, with their inherent high sensitivities, fast response times and simple equipment, has already provided several electrochemical aptamer-based sensors. Moreover, the small size and versatility of aptamers allow efficient immobilisations in high-density monolayers, an important feature towards miniaturisation and integration of compact electrochemical devices. This review describes the state-of-the-art of electrochemical aptamer-based sensors, entering into the details of the different strategies and types of electrochemical transduction and also considering their advantages when applied to the analysis of complex matrices.     

Clinical aspects of radiolabeled aptamers in diagnostic nuclear medicine: A new class of targeted radiopharmaceuticals

Targeted radiopharmaceuticals offer the possibility of improved imaging with reduced side effects. Up to now, a variety of biological receptors such as aptamers have been successfully radiolabeled and applied to diagnostic imaging of cancers. The concept of using radio-labeled aptamers for binding to their targets has stimulated an immense body of research in diagnostic nuclear medicine. These biological recognition elements are single-stranded oligonucleotides that interact with their target molecules with high affinity and specificity in unique three-dimensional structures. Because of their high affinity and specificity, the receptor-binding aptamers labeled with gamma emitters such as ^99mTc, ⁶⁴Cu, ¹¹¹In, ¹⁸F and ⁶⁷Ga can facilitate the visualization of receptor-expressing tissues noninvasively. Compared to the antibody-based radiopharmaceuticals, the radiolabeled aptamers provide a number of advantages for clinical diagnostics including high stability, low cost, and ease of production and modification, low immunogenicity and, especially, superior tissue penetration because of their smaller size. In this review, we present recent progresses and challenges in aptamer-based diagnostic radiopharmaceuticals and highlight some representative applications of aptamers in nuclear medicine.     

CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface -Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.     

Assays for aptamer-based platforms

Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.     

DNA aptamers for as analytical tools for the quantitative analysis of DNA-dealkylating enzymes

The AlkB family of oxygenases catalyze the removal of alkyl groups from nucleic acid substrates in an iron and 2-oxoglutarate-dependent manner and have roles including in DNA repair. To understand the biological functions of these DNA-dealkylating enzymes it is desirable to measure their expression levels in vitro and in vivo in complex biological matrixes. Quantitative analyses of the enzymes require affinity probes capable of binding AlkB family members selectively and with high affinity. Here we report that DNA aptamers can serve as efficient affinity probes for quantitative detection of such enzymes in vitro. Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) was applied as a general tool for: (i) selection of DNA aptamers, (ii) characterization of binding parameters for the aptamers, and (iii) quantitative detection of the target in an aptamer-based affinity analysis. The selected aptamers have a range of K_d values between 20 and 240 nM. The aptamers enabled accurate quantitative analysis of AlkB even in the presence of the Escherichia coli cell lysate. Aptamers can likely be developed for other nucleic acid repair enzymes. They may also be developed for use in in vitro and potentially in vivo studies of known nucleic acid-modifying enzymes including for functional analysis.     

Aptamer-based depletion of small molecular contaminants: A case study using ochratoxin A

Based on the increasing demand for detection and depletion of small molecules like mycotoxins or pesticides in food, water, or pharmaceuticals, aptamers are gaining more importance as sensitive, specific-depletion molecules. Here, we present an aptamer-based method for depletion of ochratoxin A (OTA) as a model system and show the advantages and the limitations of aptamers in the depletion of small molecular contaminants. OTA is a mycotoxin produced by various Penicillium and Aspergillus strains and is often found in grain and grain derivatives. We immobilized a well-described DNA aptamer against OTA on an agarose gel and used the column as a clean-up system. The aptamer shows a high specificity and sensitivity for OTA: Ochratoxin B, a molecule similar to OTA, was not bound by the aptamer; and a control oligonucleotide was not able to bind OTA. After optimizing the process for better economic feasibility, the column could be used for several times without loss of aptamer activity. We investigated the location of immobilized aptamer within the gel using fluorescent-labeled aptamers. Furthermore, beer samples spiked with OTA were used to investigate aptamer activity in complex samples. Using these complex samples we have observed a significant loss of aptamer activity. We have further investigated this limitation by performing microscale thermophoresis experiments to determine the KD values of the aptamer in different complex samples like beer, coffee, juice and wine. Our results indicate that the applicability of aptamers to real processes is currently restricted by the selection buffer used during its selection process (SELEX). We therefore suggest using conditions closer to those of the later application of the aptamer during future SELEX experiments.     

Selection of DNA aptamers against polychlorinated biphenyls as potential biorecognition elements for environmental analysis

Polychlorinated biphenyls (PCBs) have been of major concerns for decades due to their potential toxicity to human health. To trace the PCBs efficiently and sensitively, many detection methods have been developed. Aptamers, a new class of diagnostic tools, are considered to be such additional candidates for detection of pollutants. In the current study, we report the DNA aptamers, isolated by FluMag-SELEX (a modified SELEX [systematic evolution of ligands by exponential enrichment] technology), that recognize PCBs with the dissociation constants (Kd values) down to the micromolar range. Using the selected aptamers, a highly sensitive aptamer-based fluorescent assay for detection of PCBs was established using gold nanoparticles, with a widely linear range from 0.1 to 100 ng/ml. Moreover, our aptamer-based gold nanoprobe displays specificity toward 3,3',4,4'-tetrachlorobiphenyl (PCB77) compared with a few common PCB77 structural analogs. These results open the possibility of using aptamers as biorecognition elements for easy and fast environmental monitoring.     

Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin

Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant (K_d) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL⁻¹, and the limit of detection was found to be 48 pg mL⁻¹. The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.     

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands

SELEX stands for systematic evolution of ligands by exponential enrichment. This method, described primarily in 1990 [Ellington, A.D., Szostak, J.W., 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818-822; Tuerk, C., Gold, L., 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505-510] aims at the development of aptamers, which are oligonucleotides (RNA or ssDNA) binding to their target with high selectivity and sensitivity because of their three-dimensional shape. Aptamers are all new ligands with a high affinity for considerably differing molecules ranging from large targets as proteins over peptides, complex molecules to drugs and organic small molecules or even metal ions. Aptamers are widely used, including medical and pharmaceutical basic research, drug development, diagnosis, and therapy. Analytical and separation tools bearing aptamers as molecular recognition and binding elements are another big field of application. Moreover, aptamers are used for the investigation of binding phenomena in proteomics. The SELEX method was modified over the years in different ways to become more efficient and less time consuming, to reach higher affinities of the aptamers selected and for automation of the process. This review is focused on the development of aptamers by use of SELEX and gives an overview about technologies, advantages, limitations, and applications of aptamers.     

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.     

适配体与病毒性感染诊断及治疗

适配体（Aptamers）是通过指数富集的配体系统进化（systematic evolution of ligands by exponential enrichment,SELEX）技术,从随机核酸文库中筛选出来的单链寡核苷酸,已在临床医疗及其他领域得到日益广泛的应用.与抗体相比,适配体具有很多优点,如高亲和力、高特异性、分子量小、几乎无免疫排斥反应、结构稳定、易于合成等.可用于适配体筛选的靶标范围非常广,包括有机小分子、蛋白、完整细胞及病毒颗粒等.迅速可靠的病原检测对于病毒性传染病的成功预防和治疗具有重要意义.随着严格筛选和快速分离技术的进步,适配体在病毒感染的检测治疗中显示出巨大的潜力.本文概括介绍了适配体在病毒研究方面的最新应用进展及未来前景.     

Aptamers: versatile probes for flow cytometry

Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.     

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.