cell-SELEX Procedure Research Articles

Generation and Selection of Specific Aptamers Targeting Brucella Species through an Enhanced Cell-SELEX Methodology.

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the Brucella group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole Brucella cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (KD) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors’ best knowledge.

Identification of DNA aptamers that specifically targets EBV+ nasopharyngeal carcinoma via binding with EphA2/CD98hc complex

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies and has a distinct geographical distribution. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for biomarkers for early diagnosis and effective treatments. In this study, we developed DNA aptamers that specifically bind to EBV positive NPC cells by the Cell-SELEX procedure. We further identified the EphA2 (ephrin type-A receptor 2)/CD98hc (CD98 heavy chain) complex as the potential target of the aptamer EA-3 by combining aptamer-based separation and mass spectrometry analysis. Our results revealed for the first time that EphA2 colocalized with CD98hc at the plasma membrane and EphA2 coimmunoprecipitated with CD98hc, which may serve as a starting point for exploring the potential functions of the complex of EphA2 and CD98hc in NPCs. Here, we demonstrated that aptamers can be useful for the identification of protein complexes on the surface of cancer cells.

Systematic evaluation of cell-SELEX enriched aptamers binding to breast cancer cells

The sensitive and specific detection of pathogenic cells is essential in clinical diagnostics. To achieve this, molecular tools are required that unequivocally recognise appropriate cell surface molecules, such as biomarkers that come along with disease onset and progression. Aptamers are short single-stranded oligonucleotides that interact with cognate target molecules with high affinity and specificity. Within the last years they have gained an increased attention as cell-recognition tools. Here, we report a systematic analysis of a cell-SELEX procedure, for the identification of aptamers that recognise breast cancer cells. Besides a comparison of conventional (Sanger) with high-throughput sequencing techniques (next-generation sequencing), three different screening techniques have been applied to characterise the binding properties of selected aptamer candidates. This method has been found to be beneficial in finding DNA aptamers, rarely enriched in the libraries. Finally, four DNA aptamers were identified that exhibit broad-spectrum interaction patterns to different cancer cell lines derived from solid tumours.

DNA aptamer identification and characterization for E. coli O157 detection using cell based SELEX method

Escherichia coli (E. coli) O157:H7 is a foodborne pathogen that causes symptoms in humans. Its rapid identification should be considered to avoid toxic effects of the pathogen.In this study, systematic evolution of ligands by exponential enrichment using whole cells (Cell-SELEX) method was used for recognizing E. coli strain, O157 by single-stranded DNA library of aptamer. Nine rounds of cell-selex procedure were applied using O157, as a whole-cell target, with O42, K12, Top10, DH5α E. coli cells, Shigella flexneri and Salmonella typhi as counterparts. The specific interaction between selected DNA aptamers and targeted cell was assessed. After applying six rounds of SELEX for selection of DNA aptamers, the candidate sequences were obtained. Finally, specific aptamer was selected as an ideal aptamer for detection and capturing of E. coli O157. Dissociation constant of the selected aptamer were calculated (107.6 ± 67.8 pM). In addition, the secondary structure prediction and cross reactivity assays were performed. The isolated aptamer efficiency was confirmed and it was shown that the new DNA aptamer sequence has the ability to use for detection. This specific O157:H7 aptamer have the potential for application as a diagnostic ligand and could be used for detection of the related food borne diseases.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5′-end also contributes essentially to the aptameric function.

CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX.

Nasopharyngeal carcinoma (NPC) is one of the most prevailing cancers in southern China and southern Asia. Because of the nonspecific symptoms and lack of effective biomarker, most patients are diagnosed at advanced stages, resulting in poor 5-year survival rate. To identify a novel NPC biomarker facilitating early detection and effective therapy of NPC, a two-step strategy consisting of cancer cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) procedure and aptamer-based purification approach was developed. Using cell-SELEX procedure, four aptamers (S3, S5, S12 and S27) differentiating the molecular differences between NPC cells and NP cells were successfully screened. Then, using aptamer-based protein purification, membrane protein CD109 was identified as the target of aptamer S3. CD109 protein was further identified to be over-expressed in NPC cell lines and clinic tissues, but not or low in NP cell line and clinic NP tissues, detected by western blot and immunohistochemistry experiments. Our study demonstrated that CD109 identified by cell-SELEX and aptamer-based purification strategy might be used as a potential NPC biomarker for early diagnosis and targeted therapy.

Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line

Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus being unable to predict the clinical outcome. Therefore, searching for new biomarkers aiming to improve diagnosis, prognosis and therapy is still required. In this study, we performed 3D Cell-SELEX against PC-3 prostate cancer cell line, a novel strategy to select specific nucleic acid ligands against spheroid cells in 3D cell culture. This original system combines Cell-SELEX, a process that exploits the cellular structure to generate specific ligands, and 3D cell culture, an approach that mimics the tissue microenvironment in vitro. In the first round of 3D Cell-SELEX, a negative selection against RWPE-1, non-tumor cell line, was performed to subtract non-tumor specific aptamers. The supernatant was used in eight additional rounds of selection, which were performed against PC-3 cell line. After nine selection cycles, eight PC-3 specific RNA aptamers were selected and sequenced. The aptamers presented sizes between 20 and 50 nucleotides-long, with low free energy (∆G<−13.6), which contributed for their spontaneous folding and high stability. Furthermore, our results showed the aptamer A4 as a specific ligand to prostate tumor cells, with dissociation constant in the nanomolar scale. Therefore, the novel 3D Cell-SELEX procedure improved the selection of PCa cell-surface ligands and the aptamer A4 has shown potential for the identification of prostate tumor cells, suggesting the application of this molecule in further screening assays for PCa.

Microfluidic chip system for the selection and enrichment of cell binding aptamers.

Aptamers are promising cell targeting ligands for several applications such as for the diagnosis, therapy, and drug delivery. Especially, in the field of regenerative medicine, stem cell specific aptamers have an enormous potential. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by Exponential enrichment), aptamers are selected from a huge oligonucleotide library consisting of approximately 10(15) different oligonucleotides. Here, we developed a microfluidic chip system that can be used for the selection of cell specific aptamers. The major drawbacks of common cell-SELEX methods are the inefficient elimination of the unspecifically bound oligonucleotides from the cell surface and the unspecific binding/uptake of oligonucleotides by dead cells. To overcome these obstacles, a microfluidic device, which enables the simultaneous performance of dielectrophoresis and electrophoresis in the same device, was designed. Using this system, viable cells can be selectively assembled by dielectrophoresis between the electrodes and then incubated with the oligonucleotides. To reduce the rate of unspecifically bound sequences, electrophoretic fields can be applied in order to draw loosely bound oligonucleotides away from the cells. Furthermore, by increasing the flow rate in the chip during the iterative rounds of SELEX, the selection pressure can be improved and aptamers with higher affinities and specificities can be obtained. This new microfluidic device has a tremendous capability to improve the cell-SELEX procedure and to select highly specific aptamers.

Development of an Efficient Targeted Cell-SELEX Procedure for DNA Aptamer Reagents

BackgroundDNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification.MethodologyWe modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit+ hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types.Conclusions/SignificanceHere we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).

Cell-SELEX based selection and characterization of DNA aptamer recognizing human hepatocarcinoma

Single-stranded DNA aptamers recognizing human hepatocarcinoma were isolated by means of a systematic evolution of ligands by exponential enrichment using whole cells as targets (cell-SELEX). After 11 rounds of cell-SELEX procedure using human hepatoma HepG2 cells as targets and human normal hepatocyte cells as counterparts, 12 independent DNA aptamer candidate sequences were obtained. The specific interaction between selected DNA aptamers and targeted cell was confirmed. Dissociation constants of the 12 sequences obtained were also estimated in the range of 19–450nM. Moreover, the consensus secondary structure was found in the isolated aptamers, which was responsible to the recognition of HepG2 cells.

Generation of an enriched pool of DNA aptamers for an HER2‐overexpressing cell line selected by Cell SELEX

Overexpression of human epidermal growth factor receptor 2 (HER2) occurs in a large percentage of breast cancers. Monoclonal antibodies targeting HER2 are vastly used for both diagnostic and therapeutic aims. However, identifying a new molecular probe against HER2 with improved diagnostic and therapeutic features is of great importance. In this report, we have applied the cell systematic evolution of ligands by exponential enrichment (SELEX) strategy for 16 selection rounds to generate an enriched pool of aptamers that specifically recognize the HER2 positive cell line. During the Cell SELEX procedure, a human HER2-overexpressing breast cancer cell line and a human HER2 negative breast cancer cell line were used. Our results reveal that polymerase chain reaction (PCR) amplification of random DNA libraries and the selected single-stranded DNA pool in different Cell SELEX rounds are different from what we expect from PCR amplification of homologous DNA. Our results also confirmed previous studies describing positive HER2 status of SK-BR3 and the absence of the HER2 expression in the MDA-MB468. We also developed a new method, Cell enzyme-linked assay, to monitor the enrichment of aptamers in a given round of Cell SELEX. This method would also be useful in other experiments using live cell enzyme-linked immunosorbent assay on adherent cells.

Generating Aptamers for Recognition of Virus-Infected Cells

The development of molecular probes capable of recognizing virus-infected cells is essential to meet the serious clinical, therapeutic, and national-security challenges confronting virology today. We report the development of DNA aptamers as probes for the selective targeting of virus-infected living cells. To create aptamer probes capable of recognizing virus-infected cells, we used cell-SELEX (systematic evolution of ligands via exponential enrichment), which uses intact infected live cells as targets for aptamer selection. In this study, vaccinia virus-infected and -uninfected lung cancer A549 cells were chosen to develop our model probes. A panel of aptamers has been evolved by means of the infected cell-SELEX procedure. The results demonstrate that the aptamers bind selectively to vaccinia virus-infected A549 cells with apparent equilibrium dissociation constants in the nanomolar range. In addition, these aptamers can specifically recognize a variety of target infected cell lines. The aptamers' target is most likely a viral protein located on the cell surface. The success of developing a panel of DNA-aptamer probes capable of recognizing virus-infected cells via a whole living cell-SELEX selection strategy may increase our understanding of the molecular signatures of infected cells. Our findings suggest that aptamers can be developed as molecular probes for use as diagnostic and therapeutic reagents and for facilitating drug delivery against infected cells.