ssDNA Aptamer Research Articles

An ultra-sensitive detection of a whole virus using dual aptamers developed by immobilization-free screening

In this study, we successfully developed a ssDNA aptamer pairs by using an advanced immobilization-free SELEX method with affinity-based selection and counter-screening process at every round. By implementing this method, two different aptamers specifically binding to bovine viral diarrhea virus type 1(BVDV type 1) with high affinity were successfully screened. This aptamer pair was applied to ultrasensitive detection platform for BVDV type 1 in a sandwich manner. The ultrasensitive detection of BVDV type 1 using one of aptamers conjugated with gold nanoparticles was obtained in aptamer–aptamer sandwich type sensing format, with the limit of detection of 800copies/ml, which is comparable to a real-time PCR method.

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 (Kd 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.

Selection and identification of ssDNA aptamers recognizing zearalenone

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 (K(d)) 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.

Abstract 4337: Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing.

Abstract The mucin, MUC16, facilitates metastasis and immuneprotection of ovarian tumors. Therefore, agents that effectively target MUC16 expressing ovarian cancer cells will be useful for diagnostic and therapeutic applications. Here, we have utilized the Cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) approach to generate high affinity single-stranded DNA (ssDNA) aptamers as agents for detection and treatment of MUC16-positive ovarian tumors. An ssDNA aptamer library composed of ∼100 trillion sequences was subjected to ten iterative rounds of negative and positive selection using the MUC16-knockdown and MUC16-positive ovarian cancer cell line, OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high throughput NextGen sequencing. Eight ssDNA aptamers enriched through the selection process were identified by bioinformatics analysis and their selectivity and affinity for MUC16-positive cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to the MUC16-positive OVCAR-3 with a Kd of 24 and 28 nM, respectively. In contrast, Apt-1 and Apt-8 only weakly recognized the MUC16-knockdown OVCAR-3 cells. M-Fold analysis indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of NextGen sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an exhaustive library of ssDNA sequences. This study provides a streamlined method for development of theranostic aptamers that can be used to recognize and target MUC16 expressing ovarian cancer cells. Citation Format: Rebecca J. Whelan, Arvinder Kapur, Mildred Felder, Jeff Nie, Manish S. Patankar. Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2013-4337

Development of ssDNA‐based aptamers specific to PSMA

Prostate‐specific membrane antigen (PSMA) has been considered a valuable biomarker for specific imaging detection and selective targeted therapy of prostate cancer. Compared to antibodies, the aptamers, a new class of molecular probes, have more flexibility, less blood resistance and much less immunogenicity in vivo. However, the biostability of RNA‐based aptamers for PSMA is still a major concern for in vivo use. So we employed a unique SELEX approach by combination of both cell‐based selection and biomarker‐based enrichment to develop ssDNA aptamers specific to PSMA. The cultured PSMA‐positive prostate cancer cells LNCaP was used for cell‐SELEX. After 10 rounds of positive cell selection and 6 rounds of counter‐selection, the enriched ssDNA aptamers were labeled with Cy5 fluorescent reporter and their cell binding capacity was evaluated. Flow cytometry analysis revealed that 84% of LNCaP cells were highlighted by the enriched ssDNA aptamers at the concentration of 50nM, similar to that stained by anti‐PSMA antibodies. In control experiments, no binding of the enriched ssDNA aptamers to the PSMA‐negative cancer cells were observed under the same condition. Sequence analysis and sensitivity/specificity assays of the enriched aptamers are currently undergoing. The results will be presented at the meeting. Supported by NIH grants R01 CA151955.

Aptamer‐Drug Conjugates for Targeted Therapy of CD30‐expressing Lymphomas

CD30 is a biomarker for diagnosis and targeted therapy of Anaplastic Large cell lymphoma (ALCL) and Classical Hodgkin Lymphoma (CHL). Our previous studies have demonstrated specific binding of RNA aptamer to CD30 positive tumor cells. However, for in vivo targeted therapy there is a concern about biostability of the RNA aptamers.To overcome this, an ssDNA aptamer was developed via a hybrid SELEX approach using CD30‐expresing lymphoma cells and purified CD30 protein to yield the aptamer C2NP which specifically bound to CD30 expressing ALCL and CHL cells at concentrations ≤10 nm and did not react to control tumor cells. Importantly, C2NP is highly stable and more than 50% of them remained after incubation in human serum at 37°C for 24 hours, indicating the suitability for in vivo use.For targeted therapy study, C2NP was conjugated with a chemotherapeutic agent and a fluorescent reporter to form an aptamer‐drug conjugate (ADC). The specificity of ADC was confirmed by flow cytometry and intracellular delivery of chemo‐drug was observed by microscopy. Chemical conjugation of drug had no effect on aptamer binding specificity and affinity to lymphoma cells. In addition, potential therapeutic effects of the aptamer‐drug conjugate are under investigation by using cultured ALCL and CHL cell‐lines currently. This is the first study to use an aptamer‐drug conjugate for targeted therapy of CD30‐exprssing lymphomas.

Novel Design of High Affinity Beta‐Lactamase Inhibitors

Resistance to antibiotics is a major public health care concern. Most of the broad spectrum antibiotics such as penicillin's, could be inactivated in the presence of bacterial beta‐lactamases that hydrolyse the beta‐lactam ring of antibiotics resulting in the loss of antibacterial properties. Thus, blocking bacterial beta‐lactamase activity will overcome bacteria resistance to antibiotics. To develop new beta‐lactamase inhibitors we employed aptamer technology and performed SELEX enrichment of ssDNA aptamers for beta‐lactamases. Among the beta‐lactamases, CTX and KPC show the highest resistance not only to the broad spectrum antibiotics but also the newer cephalosporins and carbpenemases, and thus they were used as the targets. The SELEX was carried out using random ssDNA library with 1014–1015 unique sequences and the aptamers were enriched based on the affinity towards the beta‐lactamases. This was followed by a functional selection wherein we partitioned the progressive pools based on their inhibitory activity visualized by a Nitrocefin assay. This led us to an aptamer pool with high affinity and inhibitory activity against CTX and KPC. The obtained aptamer pool will be sequenced and the dominant sequences will be synthesized and further validated for their inhibitory capacity against various beta‐lactamases. This study opens a new direction for antibiotics development by using aptamer technology.

Selection and Characterization of Aptamers against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX)

In this paper, a high-affinity ssDNA aptamer binding to Salmonella typhimurium was obtained by a whole-bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. After nine rounds of selection with S. typhimurium as the target, a highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homology and secondary structure similarity. Eleven sequences from different families were selected for further characterization via flow cytometry analysis. The results showed that the sequence ST2P demonstrates affinity for S. typhimurium much more strongly and specifically than other sequences tested. The estimated Kd value of this particularly promising aptamer was 6.33 ± 0.58 nM. To demonstrate the potential use of the aptamers in the quantitative determination of S. typhimurium, a fluorescent bioassay with the aptamer ST2P was prepared. Under optimal conditions, the correlation between the concentration of S. typhimurium and fluorescent signal was found to be linear within the range of 50-10(6) cfu/mL (R(2) = 0.9957). The limit of detection (LOD) of the developed method was found to be 25 cfu/mL. This work demonstrates that this aptamer could potentially be used to improve the detection of S. typhimurium.

Selection, identification and application of a DNA aptamer against Listeria monocytogenes

Listeria monocytogenes is a pathogenic bacterium capable of causing severe disease leading to high hospitalization and case fatality rates. Here, we report the use of Systemic Evolution of Ligands by Exponential Enrichment (SELEX) to screen DNA aptamers that recognize L. monocytogenes with high affinity and specificity. Whole L. monocytogenes were incubated with a library of single-stranded DNA (ssDNA) aptamers. The aptamers that bound to L. monocytogenes were isolated using centrifugation, PCR amplified, and purified to yield an enriched ssDNA pool suitable for further rounds of selection. Aptamers with a dissociation constant (Kd) of 48.74 ± 3.11 nM were isolated after eight rounds of selection. An aptamer-based fluorescent bioassay was used to examine the binding ability of the selected aptamers to L. monocytogenes. To demonstrate the potential use of the aptamers in the quantitative determination of L. monocytogenes, a sandwich-type structure fluorescent bioassay with the aptamer A 15 was developed. The assay had a limit of detection (LOD) of 75 cfu/mL and a wide linear range from 102 to 107 with a correlation coefficient of 0.9937. We believe that the design of aptamer-based molecular probes that do not require the labour-intensive steps of isolating and purifying complex markers or targets represents a potentially useful method in the rapid screening and detection of foodborne pathogens.

Selection and identification of streptomycin-specific single-stranded DNA aptamers and the application in the detection of streptomycin in honey

Single-stranded DNA (ssDNA) aptamers specific to streptomycin were screened and identified from a random oligonucleotides library by affinity magnetic beads-based SELEX. After eight rounds of selection, 16 ssDNA with different sequences were identified. Then the dissociation constants (Kd) of these ssDNA were determined and an aptamer (STR1) with highest affinity for streptomycin was identified. Further study showed that aptamer STR1 exhibits very low affinity for other aminoglycoside antibiotics, indicating high specificity. With this aptamer, detection of streptomycin was achieved by using gold nanoparticles (AuNPs)-based colorimetric method. In the presence of streptomycin, the competitive binding of the target and the aptamer decreases the stability of AuNPs in NaCl solution, triggers the aggregation, and exhibits visible color change of AuNPs solution. Through UV–visible spectroscopic quantitative analysis, streptomycin can be detected in the range of 0.2–1.2μM. The presence of other aminoglycoside antibiotics shows neglectable disturbance. Furthermore, the established method was utilized to detect streptomycin in honey, and the same low detection limit and linear detection range were achieved.

Tumor Marker Detection by Aptamer-Functionalized Graphene Oxide

Interaction of the single-stranded DNA (ssDNA) aptamers and proteins was investigated on graphene oxide (GO)-based biosensors. GO was applied for biological applications because of its unique properties, which include water dispensability, exclusive selection of ssDNA, and fluorescence-quenching effects. In this study, aptamer-functionalized GO sheets of nanometer-scale thickness and micrometer-scale dimension were developed. Based on a simple mixed-and-measured strategy, platelet-derived growth factors (PDGF), the tumor marker of ovarian cancer, triggered aptamers conformation switched and deliberated aptamers from GO surface according to thermal dynamic analysis. By functionalizing with various aptamers on GO, the strategy could provide designed functions on GO-based biosensors, bioreactors, and other devices. Keywords: Aptamers, Biosensors, Functionalization, Graphene oxide, Platelet-derived growth factors, Tumor marker

DNAzyme Footprinting: Detecting Protein–Aptamer Complexation on Surfaces by Blocking DNAzyme Cleavage Activity

A novel method to quantitatively measure the binding of proteins to single-stranded DNA (ssDNA) aptamers that employs the inhibition of the DNAzyme hydrolysis of aptamer monolayers is described. A 28-base DNAzyme was designed to specifically bind to and cleave a 29-base ssDNA sequence that can fold into a G-quartet aptamer and bind the protein thrombin. The binding strength of the DNAzyme to the aptamer sequence was designed to be less than the binding strength of the thrombin to the aptamer (ΔG° = -43.1 and -51.8 kJ/mol, respectively). Formation of the thrombin-aptamer complex was found to block DNAzyme cleavage activity both in solution and in an ssDNA aptamer monolayer. We denote this method for detecting protein-aptamer complexation as "DNAzyme footprinting" in analogy to the process of DNase footprinting for the detection of protein-DNA interactions. By attaching a 40-base reporter sequence to the ssDNA aptamer monolayer, the detection of any protein-aptamer complexes remaining on the surface after DNAzyme activity can be greatly enhanced (down to one thrombin-aptamer complex per 10,000 ssDNA molecules corresponding to 100 fM thrombin in solution) by a subsequent surface RNA transcription amplification reaction followed by RNA detection with nanoparticle-enhanced SPR imaging. In addition to RNA transcription, DNAzyme footprinting can be coupled to a wide variety of other nucleic acid surface amplification schemes and thus is a powerful new route for the enzymatically amplified detection of proteins via protein-aptamer complex formation.

Isolation and characterization of DNA aptamers against Escherichia coli using a bacterial cell–systematic evolution of ligands by exponential enrichment approach

Aptamers are powerful capturing probes against various targets such as proteins, small organic compounds, metal ions, and even cells. In this study, we isolated and characterized single-stranded DNA (ssDNA) aptamers against Escherichia coli. A total of 28 ssDNAs were isolated after 10 rounds of selection using a bacterial cell–SELEX (systematic evolution of ligands by exponential enrichment) process. Other bacterial species (Klebsiella pneumoniae, Citrobacter freundii, Enterobacter aerogenes, and Staphylococcus epidermidis) were used for counter selection to enhance the selectivity of ssDNA aptamers against E. coli. Finally, four ssDNA aptamers showed high affinity and selectivity to E. coli, The dissociation constants (Kd) of these four ssDNA aptamers to E. coli were estimated to range from 12.4 to 25.2nM. These aptamers did not bind to other bacterial species, including four counter cells, but they showed affinity to different E. coli strains. The binding of these four aptamers to E. coli was observed directly by fluorescence microscopy.

Aptamer‐Assisted Gold Nanoparticles/PEDOT Platform for Ultrasensitive Detection of LPS

AbstractNeatly arranged gold nanoparticles (AuNPs) were directly electrodeposited on an electrochemically polymerized self‐assembled monolayer (SAM) of thiol‐functionalized 3,4‐ethylenedioxythiophene (EDOT) derivative, EDTMSHA. A thiolated single‐stranded DNA (ssDNA) aptamer with high specificity to LPS was immobilized on the AuNPs/conducting polymer composite film, serving as sensing platform for LPS detection. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscope (SEM), and atomic force microscopy (AFM) were utilized to characterize the modification and detection processes. The electron transfer resistance was found to have a linear relationship with LPS concentration from 0.1 pg/mL to 1 ng/mL.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.

Carbon nanotube decorated magnetic microspheres as an affinity matrix for biomolecules.

Carbon nanotube (CNT) decorated magnetic microspheres were fabricated to develop a multimodal platform that utilizes non-covalent molecular interactions of CNTs to magnetically separate biomolecules. Hybrid CNT-microspheres prepared by a feasible method reported herein had a well-defined structure as characterized by Raman spectroscopy and scanning electron microscopy. Binding interactions of resulting magnetic CNT-microspheres with DNA oligonucleotides were studied to demonstrate that single stranded DNA (ssDNA) in a solution can be effectively recovered by magnetic CNT-microspheres through strong physical wrapping of DNA around CNTs' walls. The magnetic character of these CNT-microspheres combined with their capability to bind other molecules including DNA allows their use as an affinity matrix that can be utilized in affinity separation of biomolecules, and also as a platform to monitor non-covalent binding interactions of CNTs with other biomolecules. As a proof of concept, we report on the use of these CNT-microspheres in in vitro selection of ssDNA aptamers against carcinoembryonic antigen (CEA), a cancer biomarker, by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). ssDNA aptamer candidates that have strong affinity towards CEA were successfully separated magnetically from a pool of ssDNA (∼1014 molecules). Our results demonstrate that CNT-microspheres can serve as strong tools for affinity separation methodologies and can be utilized for various affinity pairs in solution.

3P310 細胞表面特異的結合DNAアプタマーの作製と心筋細胞の精製(28.バイオエンジニアリング,ポスター,日本生物物理学会年会第51回(2013年度))

3P310 細胞表面特異的結合DNAアプタマーの作製と心筋細胞の精製(28.バイオエンジニアリング,ポスター,日本生物物理学会年会第51回(2013年度))

Cellular Internalization and Cytotoxicity of Aptamers Selected from Lung Cancer Cell

In this work, single-stranded DNA (ssDNA) aptamers against EGFR-transfected A549 cells, one type of non-small cell lung cancer (NSCLC) cells, were selected by cell-SELEX (systematic evolution of ligands by exponential enrichment) and were evaluated. The selected aptamers had high affinity to the A549 cells with dissociation constants in the nanomolar rang. Moreover, the aptamers were able to internalize into the cells, which is advantageous over most of the other existing aptamers. One of the selected aptamers showed significant cytotoxicity by inhibiting the cell proliferation and inducing cell apoptosis. These aptamers are expected to be new molecular probes for cancer cell targeting and drug delivery.

Function of ssDNA aptamer and aptamer pool against Mycobacterium tuberculosis in a mouse model

Novel antibacterial agents against Mycobacterium tuberculosis (MTB) are crucial due to the high infection and mortality rates associated with the disease. Our previous study confirmed that aptamers from a whole bacterium obtained by the Systematic Evolution of Ligands by Exponential Enrichment method specifically bound to the MTB virulent strain (H37Rv). In the present study, the function of aptamers against MTB in a mouse model was further determined. It was demonstrated that the NK2 aptamer has marked inhibitory effects on the adhesion/invasion of H37Rv to macrophages in vitro and stimulates intracellular IFN-γ production in CD4+ T-cells. The aptamer pool exhibited the strongest inhibitory effect on H37Rv adhesion/invasion to CD8+ T-cells in vitro compared with all aptamers-treated and control groups. Histopathological examination of lung biopsy specimens revealed a correlation between aptamer presence and lower pulmonary alveoli fusion, swelling and more prominent air spaces. Acid-fast staining of biopsy specimens from the lungs of the mice demonstrated parallel treatment effects. Results of the present study indicate that the 10th pool aptamers and NK2 may play an active role against H37Rv, however, the effect was different in vivo and in vitro. The treatment effect of 10th pool aptamers was found to be better in comparison to NK2 in vivo. Additional target sites involved in pathogenicity of H37Rv were also revealed and the NK2 binding site and aptamers, including the 10th pool aptamers, may antagonize these sites. Further studies are required to screen for other valuable aptamers which may be used as therapeutic drugs in combination with NK2.

Detection of VR-2332 Strain of Porcine Reproductive and Respiratory Syndrome Virus Type II Using an Aptamer-Based Sandwich-Type Assay

Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome disease (PRRS), a disease that has a significant and economic impact on the swine industry. In this study, single-stranded DNA (ssDNA) aptamers with high specificity and affinity against VR-2332 strain of PRRSV type II were successfully obtained. Of 19 candidates, the LB32 aptamer was found to be the most specific and sensitive to VR-2332 strain according to an aptamer-based surface plasmon resonance (SPR) analysis. The detection of VR-2332 of PRRSV type II was successfully accomplished using the enzyme-linked antibody-aptamer sandwich (ELAAS) method. The detection limit of ELAAS was 4.8 × 10(0) TCID(50)/mL that is comparable to some of the previous reports of the PCR-based detection but does not require any complicated equipment or extra costs. Moreover, this ELAAS-based PRRSV detection showed similar sensitivity for both the VR-2332 samples spiked in diluted swine serum and in buffer. Therefore, this VR-2332 strain-specific aptamer and its assay method with high specificity can be used as an alternative method for the fast and precise detection of PRRSV.