Single-stranded DNA aptamers that bind differentiated but not parental cells: subtractive systematic evolution of ligands by exponential enrichment

In this paper, single-stranded (ss)DNA aptamers with capability to distinguish differentiated PC12 cells from normal PC12 cells were selected by subtractive systematic evolution of ligands by exponential enrichment (SELEX) method. Before each round of selection, randomized ssDNAs were incubated with regular PC12 cells to eliminate those that recognize the common cellular components of both differentiated and undifferentiated PC12 cells. After six rounds of cell-based selection, both of individual aptamers and aptamers of the sixth round pool were found binding to differentiated PC12 cells, but not to the parental PC12 cells. The aptamers of the starting pool showed no such binding. Sequence analysis illustrated that the amount of G content in central random region of these aptamers was much higher than that of the starting pool, which would be expected to be average. The aptamers obtained from this method were also able to identify differentiated PC12 cells from a mixture of both normal and differentiated cells. The results indicate that subtractive SELEX is a useful tool in finding ligands to specific biological markers that distinguish a subtype of cells from cells of homologous origin, such as carcinoma cells among normal epithelial tissues. Both these aptamers and their markers may play important roles in basic research and clinical diagnosis.     

Structural characterization of an anti-gp120 RNA aptamer that neutralizes R5 strains of HIV-1

We recently described the isolation of 2'-fluoropyrimidine-substituted RNA aptamers that bind specifically to the surface glycoprotein (gp 120) of the R5 strain, HIV-1(Ba-L), as presented in a previous study. These aptamers potently neutralize HIV-1 infectivity in human peripheral blood mononuclear cells of both tissue culture lab-adapted strains and diverse R5 clinical isolates from multiple clades. Here, we report a detailed structural characterization of one such neutralizing aptamer, B40, using enzymatic and chemical probing methods. We identify the minimal region of the aptamer essential for binding gp120 and accordingly design a 77-nucleotide truncated aptamer, B40t77. We then quantitatively analyze the binding affinity and neutralization potency of the parental and truncated (minimal) aptamer, and show them to be comparable. Furthermore, using results from secondary structure analysis, RNA mutagenesis and BIAcore surface plasmon resonance (SPR) binding assays, we hypothesize that a folded RNA structure is required to present specific nucleotide sequences to allow gp120-recognition and binding. The information gained from this study may provide leads for development of novel anti-HIV-1 therapies and can be used to extend our understanding of the molecular interactions between the virus and its host cell.     

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.     

Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide 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.     

金黄色葡萄球菌外毒素B特异性适体的筛选及其应用

目的:利用指数富集配基的系统进化(SELEX)技术,筛选能与金黄色葡萄球菌外毒素B(SEB)特异、高亲和力结合的单链DNA(ssDNA)适体,并将该适体应用于患者血清标本的检测。方法:从体外合成的96核苷酸随机ss-DNA文库中,以羧基磁珠作为筛选介质,经逐步PCR扩增、筛选,获得针对SEB的高亲和力、高特异性适体;利用荧光素标记适体测定筛选过程中各轮结合力;利用酶连接适体方法检测适体特异性和结合力。结果:经过13轮筛选,ssDNA文库与SEB的结合百分率从1.1%提高到39.8%,增加了36倍;获得的ssDNA适体(A11)针对SEB的特异性强,与金黄色葡萄球菌表面蛋白A(SPA)结合低,并能初步识别患者血清。结论:利用SELEX技术筛选获得了特异结合SEB的高亲和力的ssDNA适体,为金黄色葡萄球菌的临床诊断与治疗奠定了基础。     

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called “Ligand-Guided-Selection” (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.     

Probing high affinity sequences of DNA aptamer against VEGF165

Vascular endothelial growth factor (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(121), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.     

In vitro selection of RNA aptamers that selectively bind danofloxacin

Danofloxacin is a synthetic fluoroquinolone with broad spectrum antibacterial activity that is used for the treatment of respiratory diseases in animal husbandry. However, danofloxacin has many adverse reactions and is toxic to humans. Especially, it detrimentally affects muscle, central nerve system, peripheral nerve system, liver, and skin in those who ingest foods in which danofloxacin has accumulated. Prescreening and determination of the level of danofloxacin in foods or food products is necessary for human health. Aptamers are composing of oligonucleotides that specifically interact with target molecules. They are emerging as detection/diagnostic ligands. Here, we used the SELEX in vitro selection technology to identify specific and high-affinity RNA aptamers with 2′-fluoro-2′-deoxyribonucleotide modified pyrimidine nucleotides against danofloxacin. Selected RNA aptamers bound specifically to danofloxacin, but not to tetracycline. Truncation of RNA aptamer up to 36 mer did not comprise specificity and affinity. The truncated RNA aptamer specifically bound to target chemical, allowing the discrimination of danofloxacin from other fluoroquinolones. The isolated specific aptamer could be a potential agent used for the rapid and cost-effective detection and sensing of danofloxacin, replacing instrumental methods including the more expensive and time-consuming methods of high performance liquid chromatography and liquid chromatography/mass spectrometry.     

Nucleic acid aptamers for capture and detection of Listeria spp

The purpose of this study was to identify biotinylated single-stranded (ss) DNA aptamers with binding specificity to Listeria and use these for capture and subsequent qPCR detection of the organism. For aptamer selection, SELEX (systematic evolution of ligands by exponential enrichment) was applied to a biotin-labeled ssDNA combinatorial library. After multiple rounds of selection and counter-selection, aptamers separated, sequenced, and characterized by flow cytometry showed binding affinities to L. monocytogenes of 18–23%. Although selected for using L. monocytogenes, these aptamers showed similar binding affinity for other members of the Listeria genus and low binding affinity for non-Listeria species. One aptamer, Lbi-17, was chosen for development of a prototype capture and detection assay. When Lbi-17 was conjugated to magnetic beads and used in a combined aptamer magnetic capture (AMC)-qPCR assay, the pathogen could be detected at concentrations <60 CFU/500 μl buffer in the presence of a heterogeneous cocktail of non-Listeria bacterial cells, with a capture efficiency of 26–77%. Parallel experiments using immunomagnetic separation (IMS)-qPCR produced the same detection limit but lower capture efficiency (16–21%). Increasing assay volume to 10 and 50 ml resulted in reduced capture efficiency and higher limits of detection, at 2.7 and 4.8 log₁₀ CFU L. monocytogenes per sample, respectively, for the AMC-qPCR assay. Biotinylated ssDNA aptamers are promising ligands for food-borne pathogen concentration prior to detection using molecular methods.     

两种富集方法相结合对蓖麻毒素进行SELEX筛选研究

为了获得能特异识别具有细胞毒性的蓖麻毒素蛋白寡核苷酸适配子,体外构建了含40个随机序列全长87nt的随机ssDNA文库,采用指数富集配基的系统进化(SELEX)技术方法,结合微孔板和亲和树脂两种分离、富集方法,经过数轮筛选,文库与蓖麻毒素的结合率达到了38．5％。结果表明,以亲和树脂为分离介质进行筛选,富集效果非常明显。     

NGF-differentiated and undifferentiated PC12 cells vary in induction of apoptosis by ethanol.

The present study was undertaken to determine whether the neurotoxic effects of ethanol vary between undifferentiated and differentiated neurons. For this study, untreated rat pheochromocytoma (PC12) cells and PC12 cells treated for 8-10 days with nerve growth factor (NGF) were used as models of undifferentiated and differentiated neurons, respectively. Treatment of differentiated PC12 cells with 150 mM ethanol resulted in a loss of cells whereas a similar treatment of undifferentiated cells had no effect. In contrast, 50 mM ethanol enhanced apoptosis initiated by serum withdrawal in undifferentiated cells while a similar response in the differentiated cells required 150 mM ethanol. This study demonstrates that undifferentiated and differentiated neuronal cells differ in their sensitivity to the neurotoxic actions of ethanol.     

全细菌SELEX法筛选粪肠球菌特异性适配体

肠球菌（Enterococcus）是内源性和外源性医院感染的第二大病原菌,检出率仅次于大肠杆菌,从分子水平上发展靶标的高亲和力分子探针对肠球菌的识别和检测具有非常重要的意义。本研究以粪肠球菌为靶标,运用全细菌指数富集的配体系统进化技术（whole-bacteria systematic evolution of ligands by exponential enrichment, whole-bacteria SELEX）,从全长为79个核苷酸包含35个随机碱基序列的单链DNA文库中筛选与靶标高亲和力、高特异性结合的适配体,利用荧光分析法监控筛选过程中不同轮次所得次级文库与粪肠球菌的结合力,经12轮筛选和克隆测序,获得了39条适配体序列。进一步对筛选得到的适配体进行序列比对、二级结构分析、流式细胞分析、解离常数（Kd）测定及特异性验证,最终获得一条与粪肠球菌能特异性结合的适配体Apt 21,其Kd值为549.2 ± 147.4 nmol/L。该适配体可作为粪肠球菌检测的识别元件,为建立基于适配体的新型粪肠球菌检测方法奠定了基础。     

DNA aptamers against the receptor binding region of hemagglutinin prevent avian influenza viral infection

The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular region (residues 101–257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells.     

Selection of DNA aptamers that bind to four organophosphorus pesticides

Single-stranded DNA (ssDNA) aptamers against four organophosphorus pesticides (phorate, profenofos, isocarbophos and omethoate) were simultaneously isolated from an immobilized random ssDNA library by systematic evolution of ligands by exponential enrichment (SELEX) technique. After 12 rounds of in vitro selection, five ssDNA aptamer candidates were selected and their binding affinities were identified by a novel method using a molecular beacon. Two of the five ssDNA sequences, SS2-55 and SS4-54, demonstrated higher affinities and specificities to the four organophosphorus pesticides. They were defined as broad-spectrum aptamers binding to four different targets and their simulated secondary structures showed highly distinct features with typical stem and loop structures. The dissociation constant of SS2-55 and SS4-54 binding to the four organophosphorus pesticides ranged from 0.8 to 2.5?μM. These aptamers offered application potential in the analysis and/or neutralization of the residues of the four organophosphorus pesticides.     

Selection of aptamers against inactive Vibrio alginolyticus and application in a qualitative detection assay

Aptamers against inactive Vibrio alginolyticus were selected from an 82-nt ssDNA random library by systematic evolution of ligands by exponential enrichment. After 15 rounds of selection, the final pool of aptamers was highly specific for inactivated V. alginolyticus and had a dissociation constant of 27.5 ± 9.2 nM. Using these aptamers and PCR, V. alginolyticus could be detected at 100 cells/ml. Sequencing of the final pool of aptamers revealed that some sequences, termed high-frequency aptamers, appeared more than once; these may be of practical application. All sequences obtained were divided into nine families according to their homology tree, some conserved sequences were also found in each of the six families. One sequence was found in significant proportions of the aptamers, suggesting that this conserved sequence might be important for forming the three-dimensional aptamer structure.     

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.     

Screening and Characterization of a Novel RNA Aptamer That Specifically Binds to Human Prostatic Acid Phosphatase and Human Prostate Cancer Cells

Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.