Cell-based SELEX Research Articles

New cellular RNA elimination method for cell-based SELEX of modified RNA aptamers

Aptamers are synthetic singlestranded RNA or DNA molecules that have high specificity to bind tar� get molecules by means of unique spatial structure for� mation. The method of the systematic evolution of ligands by exponential enrichment (SELEX) is used to obtain aptamers (1). Different objects can be the tar� gets for selection ranging from metal ions and small organic molecules to proteins and unicellular organ� isms. Aptamers that recognize the cells of certain type due to affinity to specific surface proteins are of great interest. As a rule, live cells are used as targets for the selection of these aptamers. The fields of application of these aptamers include the targeted delivery of drugs into cells and the specific detection of cells (2-4). Both DNA and RNA libraries are widely used to select aptamers. The advantages of RNAbased aptamers are their ability to form more diverse and stable tertiary structures than DNAbased aptamers; nonetheless, additional chemical modifications are needed to increase their biological stability. Thus, pyrimidine nucleosides within escort RNA aptamers are usually substituted by 2'�modified analogues, as a rule, by 2'�fluoroor 2'�aminonucleosides (5). A general scheme for the selection of 2'�modified RNA aptamers by cell� based SELEX method includes the following stages: (1) the incubation of the RNA library with target cells;

Using aptamers for cancer biomarker discovery.

Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment) SELEX and cell-based SELEX (cell-SELEX). Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics.

Development of a Novel DNA Aptamer Ligand Targeting to Primary Cultured Tumor Endothelial Cells by a Cell-Based SELEX Method

The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.

Generation of Lung Adenocarcinoma DNA Aptamers for Cancer Studies

Lung cancer is the most lethal malignancy in the world, and each year thousands of people die from this disease. Early detection has proven to increase the 5-year survival for this cancer in general, independent of the origination site in the lung. To address this challenge, we have used cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select a panel of aptamers capable of distinguishing lung adenocarcinoma cells from normal lung epithelial cells. These aptamers bind at physiological and formalin-fixed conditions and display affinity for their targets with apparent Kd's in the nanomolar range. Our findings suggest that the selected aptamers have the potential to be used in clinical settings, as well as to improve classification of nonsurgical specimens, another current challenge in lung cancer.

Use of Cell-SELEX to Generate DNA Aptamers as Molecular Probes of HPV-Associated Cervical Cancer Cells

BackgroundDisease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the approach used to select high affinity aptamers for specific macromolecular targets from among the >1013 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines.Methodology/Principal FindingsWhole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX)). Using this approach, we identified high affinity aptamers (nanomolar range Kd) to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa). We also performed preliminary investigation of the aptamer epitopes and their binding characteristics.Conclusions/SignificanceUsing AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of cancer biopsies and aid in the effective management of pathological conditions (diagnosis, predicted outcome, and treatment options).

Abstract 3886: Development of a new DNA aptamer against primary cultured tumor endothelial cells by a cell-based SELEX method to discover a new drug delivery system

Abstract The purpose of the present study was to develop DNA aptamers through a spontaneous cell-based SELEX method (systemic evolution of ligands by exponential enrichment) that specifically binding to cell surface proteins or biomarkers produced by primary cultured tumor endothelial cells (TECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as the development of metastasis. To combat angiogenesis, an appropriate diagnosis and understanding at the molecular level of different cancer types is now a high priority. Our initial study explored the use of the cell-based SELEX method, which has been applied in TECs to generate high affinity DNA aptamers. Our selected DNA aptamer AraHH001 bound specifically and with a high affinity to TECs in the nanomolar range but did not bind to normal skin endothelial cells (skin-ECs) and tumor parenchymal OSRC-II cells. The selected DNA aptamers also bounded to primary cultured human tumor endothelial cells (hTECs) isolated from a clinical patient with a renal carcinoma. Therefore, the development of specific DNA aptamers that bind to TECs is reported here for the first time. We conclude that such new DNA aptamers promise to hold great promise as targeted molecular probes that appear to play a major role in angiogenesis, drug delivery, identification of biomarker as well as in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3886. doi:1538-7445.AM2012-3886

Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery.

Abstract 626: Specific targeting of cancer cells with RNA aptamers

Abstract A great promise in developing specific molecular probes for disease biomarker discovery and for diagnostic and therapeutic applications is recently represented by the intact cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) strategy that allow to select nucleic acid aptamers against living cells. Indeed, using cell-SELEX to target proteins expressed on the cell surface permits to develop aptamers that are able to discriminate between even closely related target cells. This strategy has the advantage of a direct selection of ligands without prior knowledge of the target molecules and enables the identification of aptamers that bind large cell surface-specific markers, in their native conformation. Recently, by coupling a differential whole-cell SELEX protocol to human glioma and NSCLC cell lines we addressed the possibility to isolate aptamer molecules that may target cancer specific cell surface bound proteins. We isolated a set of 2’fluoro-pyrimidines modified RNA aptamers that are specific for different targets that are highly expressed on the tumor cell type used and underepresented on other cancer cell types tested. Remarkably, two of these aptamers bind at high affinity and high specificity to the extracellular domain of two human receptor tyrosine kinases (RTKs), EGFR and Axl respectively. Following binding, both aptamers hamper ligand-dependent receptor dimerization and autophosphorylation. Further, depending on the targeted RTK, the selected aptamers cause inhibition of cell proliferation and selective cell death in vitro and in vivo. Since an obvious advantage of RNA-based aptamers that bind cell surface receptors is the possibility to be conjugated to a siRNA and used as cargoes for tissue specific internalization, we thus developed a cell SELEX based protocol to enrich for aptamers that following binding to cell surface are rapidly internalized in a receptor-mediated manner. The whole-cell SELEX strategy holds a great promise in developing specific molecular probes for cancer biomarker discovery and for cancer diagnostic and therapeutic applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2011-626

Metastasis‐focused cell‐based SELEX generates aptamers inhibiting cell migration and invasion

Metastasis, the capacity of tumour cells to disseminate and grow at distant sites, is the main factor in cancer mortality. Compounds inhibiting migration and invasion of cancer cells are promising candidates for anticancer therapy strategies. We have generated nuclease-resistant RNA ligands (aptamers) recognizing highly metastatic cells with high affinity and specificity, and inhibiting their migratory and invasive potentials. Aptamers were generated by a cell-based subtractive SELEX technology using isogenic cell lines with similar tumorigenic potentials but opposite metastatic aggressiveness. Two aptamers, E37 and E10, bound specifically to the metastatically aggressive cell line and altered the phosphorylation of several tyrosine kinases. Fluorescent microscopy showed intracellular uptake of E37, in contrast to membrane binding of E10. Both aptamers inhibited migration of tumour cells in culture (50 and 85% inhibition with respect to control pool for E10 and E37, respectively) while only E10 inhibited cell invasion (-75% with respect to control pool). This proof-of-concept study demonstrates the potential of cell-based SELEX to yield ligands that selectively recognize aggressive metastatic cells and inhibit phenotypes linked to metastatic potential.

Bio-bar-code dendrimer-like DNA as signal amplifier for cancerous cells assay using ruthenium nanoparticle-based ultrasensitive chemiluminescence detection

Bio-bar-code dendrimer-like DNA (bbc-DL-DNA) is employed as a label for the amplification assay of cancer cells in combination with the newly explored chemiluminescence (CL) system of luminol-H(2)O(2)-Ru(3+) and specificity of structure-switching aptamers selected by cell-based SELEX.

Cell-based aptamer selection for diagnosing cancer and predicting cancer progression

Antibodies are excellent molecular recognition agents for a wide range of applications therefore they have been used heavily in clinical assays such as disease diagnosis. More recently, aptamers have emerged as alternative capturing agents in a variety of applications including medical diagnosis, environmental toxicity detection, targeted drug delivery and viral therapeutics. Aptamers are ssDNA or RNA that form three dimensional structures and bind to the target molecules such as peptide, protein or small molecules. Aptamers are generated by in vitro process called “SELEX (Systematic Evolution of Ligands by Exponential Enrichment)”. Conventionally, SELEX is performed with immobilized target molecules such as proteins in column, filter or beads. However, for some targets like membrane proteins, it is very difficult or almost impossible to immobilize the target proteins in their active conformation. However, cell-based aptamer selection technology explain how it can be better than standard immobilization methods in brief. Here, we described the cell-specific aptamers selecting technology, called cell-based SELEX, for diagnosing disease and predicting disease progression, especially in the case of complex disease, like cancer.

Molecular recognition of acute myeloid leukemia using aptamers

Cell surface proteins can play important roles in cancer pathogenesis. Comprehensive understanding of the surface protein expression patterns of tumor cells and, consequently, the pathogenesis of tumor cells, depends on molecular probes against these proteins. To be effectively used for tumor diagnosis, classification and therapy, such probes would be capable of specific binding to targeted tumor cells. Molecular aptamers, designer DNA/RNA probes, can address this challenge by recognizing proteins, peptides and other small molecules with high affinity and specificity. Through a process known as cell-SELEX, we used live acute myeloid leukemia (AML) cells to select a group of DNA aptamers that can recognize acute myeloid leukemia cells with dissociation constants (Kds) in the nanomolar range. Interestingly, one aptamer (KH1C12), compared with two control cell lines (K562 and NB4), showed significant selectivity to the target AML cell line (HL60) and could recognize the target cells within a complex mixture of normal bone marrow aspirates. The other two aptamers KK1B10 and KK1D04 recognize targets associated with monocytic differentiation. Our studies demonstrate that the selected aptamers can be used as a molecular tool for further understanding surface protein expression patterns on tumor cells and thus providing a foundation for effective molecular analysis of leukemia and its subcategories.

Using Aptamer–Nanoparticle Conjugates for Cancer Cells Detection

Accurate and sensitive diagnosis is particularly critical in the early stages of tumor development in order to facilitate the selection of effective therapeutic pathways and improve clinical outcomes. However, accurate and sensitive detection of malignant cells requires probes capable of differentiating the unique features of target cells at the molecular level. Recently, aptamers have emerged as a powerful class of ligands for specific biomolecular targeting. To select aptamers which can bind their target cells with high affinity and specificity, the cell-based SELEX method has been developed. Because of its fast enrichment, easy operation and ability to obtain a group of probes simultaneously, cell-based SELEX has allowed expansion of aptamer use to the fields of cancer cell detection, imaging, and targeted therapy. In large part, the development of nanomaterials as aptamer bioconjugates has increased the interest of using aptamer–nanoparticle conjugates as potential diagnostic and therapeutic vehicles. In this review, we will outline the routes of synthesis, fundamental properties, and potential applications of several types of nanoparticles, including dyedoped silica nanoparticles, gold-based nanoparticles, and magnetic nanoparticles. By combining such nanomaterials with cancer-related aptamers, a novel class of bioconjugates is emerging with promising potential for application in cancer separation, detection, imaging and targeted therapy.

CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

Aptamers Evolved from Cultured Cancer Cells Reveal Molecular Differences of Cancer Cells in Patient Samples

Molecular-level differentiation of neoplastic cells is essential for accurate and early diagnosis, but effective molecular probes for molecular analysis and profiling of neoplastic cells are not yet available. We recently developed a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy to generate aptamers (designer DNA/RNA probes) as molecular probes to recognize neoplastic cells. We tested 6 cell-SELEX-generated aptamers with equilibrium dissociation constants in the nanomolar to subnanomolar range: sgd5, selected from Toledo cells, a human diffuse large-cell lymphoma cell line (B-cell), and sgc8, sgc3, sgc4, sgd2, and sgd3 from CCRF-CEM cells, a human precursor T cell acute lymphoblastic leukemia (T-ALL) cell line. Aptamers were labeled with fluorescein isothiocyanate fluorophores and then used to recognize, by flow cytometric analysis, neoplastic cells in cultured hematopoietic cell lines and clinical samples. Aptamer sgd5 recognized only its target cells. Aptamers sgc3, sgd2, sgd3, sgc4, and sgc8, selected from a T-cell leukemia cell line, identified all of the cultured T-cell leukemia cell lines with relatively high fluorescence intensity. Aptamers sgc8, sgc3, and sgd3 showed good selectivity toward T-ALL cells and almost no binding to normal hematopoietic cells or lymphoma and myeloma cells. Selected aptamers also detected targets on the cell membranes of neoplastic cells in patient samples. Aptamers selected against cultured neoplastic cells can effectively be used as molecular probes for recognition of neoplastic cells in patient samples. Cell-based aptamer selection can be used to generate aptamer probes to obtain molecular signatures of neoplastic cells in patient samples.

Selection of Aptamers for Molecular Recognition and Characterization of Cancer Cells

In this paper, we describe a new way to generate molecular probes for specific recognition of cancer cells. Molecular medicine will require a large number of probes for molecular recognition and characterization of a variety of diseased cells. Aptamers, single-stranded DNA/RNA probes, are poised to become a chemist's antibody and have the potential to serve as molecular probes for a variety of biomedical applications. By applying newly developed cell-SELEX (cell-based systematic evolution of ligands by exponential enrichment) against whole living cells, panels of aptamers have been evolved from an initial DNA library to characterize target cells at the molecular level. Ramos cells, a B-cell lymphoma cell line, were used as target cells for the generation of effective molecular probes. By taking advantages of the repetitive and broad enrichment strategy, the selected aptamers could bind to target cells and other closely related cell lines in variant patterns with an equilibrium dissociation constant (Kd) in the nanomolar range. Some aptamers could also specifically recognize the target lymphoma cells mixed with normal human bone marrow aspirates. The cell-based SELEX is simple, fast, and robust. The strategies used here will be highly useful for aptamer selection against complex target samples in order to generate a large number of aptamers in a variety of biomedical and biotechnological applications, paving the way for molecular diagnosis, therapy, and biomarker discovery.

Aptamer-Conjugated Nanoparticles for the Collection and Detection of Multiple Cancer Cells

We have extended the use the aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells. The aptamers were selected using a cell-based SELEX strategy in our laboratory for cancer cells that, when utilized in this method, allow for the selective recognition of the cells from complex mixtures including fetal bovine serum samples. Aptamer-conjugated magnetic nanoparticles were used for the selective targeting cell extraction, and aptamer-conjugated fluorescent nanoparticles were employed for sensitive cellular detection. Employing both types of nanoparticles allows for selective and sensitive detection not possible by using the particles separately. Fluorescent nanoparticles amplify the signal intensity versus a single fluorophore label resulting in improved sensitivity. In addition, aptamer-conjugated magnetic nanoparticles allow for extraction and enrichment of target cells not possible with other separation methods. Fluorescent imaging and a microplate reader were used for cellular detection to demonstrate the wide applicability of this methodology for medical diagnostics and cell enrichment and separation.

Aptamers as potential probes for cancer

Aptamers consisting of single-stranded DNA probes have been selected to target the many surface ligands on a leukaemia T-cell line. Weihong Tan and colleagues used a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy to generate a panel of aptamers that act as probes to show the molecular differences between any two cell types. “The selected aptamers have been used for the specific recognition of leukaemia cells, but the molecular differences between target and control cells can be isolated easily, providing an effective approach to the discovery of molecular signatures of many other diseases”, explains Tan (Center for Research at the Bio/Nano Interface, University of Florida, Gainesville, FL, USA).

Aptamer-Conjugated Nanoparticles for Selective Collection and Detection of Cancer Cells

We have developed a method for the rapid collection and detection of leukemia cells using a novel two-nanoparticle assay with aptamers as the molecular recognition element. An aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for CCRF-CEM acute leukemia cells that, when applied in this method, allows for specific recognition of the cells from complex mixtures including whole blood samples. Aptamer-modified magnetic nanoparticles were used for target cell extraction, while aptamer-modified fluorescent nanoparticles were simultaneously added for sensitive cell detection. Combining two types of nanoparticles allows for rapid, selective, and sensitive detection not possible by using either particle alone. Fluorescent nanoparticles amplify the signal intensity corresponding to a single aptamer binding event, resulting in improved sensitivity over methods using individual dye-labeled probes. In addition, aptamer-modified magnetic nanoparticles allow for rapid extraction of target cells not possible with other separation methods. Fluorescent imaging and flow cytometry were used for cellular detection to demonstrate the potential application of this method for medical diagnostics.