Cell-based SELEX Research Articles

Cell-based SELEX aptamer selection for electrochemical detection of Fluoribacter bozemanae bacteria

Fluoriobacter bozemanae (F. bozemanae) can cause Legionnaires' disease. The conventional methods for the detection of F. bozemanae are laborious, costly and has to be performed in centralized labs by skilled personnel. Therefore, there is an urgent need for rapid detection methods for F. bozemanae. The present work reports the first electrochemical aptasensor for the rapid and specific detection of the most virulent type of Legionellosis. Firstly, high-affinity aptamer was selected against the F. bozemanae using cell-based systematic evolution of ligands by exponential enrichment (Cell SELEX). The aptamers selections were based on 10 rounds of cell-SELEX with live F. bozemanae cells, and the counter-selection was performed against five other closely related Legionella sub-species. The selected oligonucleotide sequences were characterized using the dissociation constant (Kd). Apt-FB6 sequence gave a Kd value as low as 9.516 nM. This selected aptamer was integrated with electrochemical impedance spectroscopy (EIS). The impedimetric aptasensor for F. bozemanae exhibited a detection limit down to 10 CFU/mL. Finally, the aptasensor was tested against Legionella anisa, Legionella micdadei, Fluoriobacter dumoffii, and Legionella pneumophila serogroup 1 and 3. The biosensor didn't show significant cross-reactivity with the closely-related sub-species of Legionella. The selected aptasensor can be further exploited for further biosensing applications for clinical, environmental, and diagnostic purposes.

Evolution of Cell-Type-Specific RNA Aptamers via Live Cell-Based SELEX.

Live cell-based SELEX (Systematic Evolution of Ligand EXponential enrichment) is a promising approach for identifying aptamers that can selectively bind to a cell-surface receptor or recognize a particular target cell population. In particular, it offers a facile selection strategy for some special cell-surface proteins that are originally glycosylated or heavily posttranslationally modified and are unavailable in their native/active conformation after in vitro expression and purification. In this chapter, we describe a generalized procedure for evolution of cell type-specific RNA aptamers targeting a cell membrane bound target by combining the live cell-based SELEX strategy with high-throughput sequencing (HTS) and bioinformatics analysis.

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19.

Simple SummaryHaematological malignancies show a constantly growing incidence, accounting for 6.5% of new cancer cases worldwide. Among them, B-cell neoplasms often show resistance to conventional chemotherapy that is also associated with numerous adverse effects. Therefore, in order for the treatment outcome to be improved, the development of new safe and effective targeted therapeutic approaches represents a main challenge. In this regard, nucleic acid aptamers are very attractive molecules. Indeed, they show high affinity and specificity for their target, increased tumour penetration, and low toxicity. Recently, CD19 has emerged as a key surface marker of malignant B cells, suitable for the development of new compounds for malignant B-cell targeting. Here, we isolated an RNA aptamer targeting the human CD19 antigen on malignant B cells that was able to rapidly internalise into target cells. Therefore, it represents a useful carrier for secondary reagents and a promising tool for the development of new safe and effective targeted therapies for B-cell malignancy treatment.The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

RNAアプタマーによる心筋細胞に特異的な細胞内薬物送達法の開発

A number of intracellular signaling proteins are involved in the progression of heart failure. Such proteins could be a potential therapeutic target for heart failure if an intracellular drug-delivery system (DDS) specific for a cardiomyocyte (CM) were achieved. To develop the CM-specific intracellular DDS, we attempted to isolate RNA aptamers which are specifically taken up by CM using the cell-based SELEX (systematic evolution of ligands by exponential enrichment) protocol with a library of RNA molecules containing a randomized 40-nucleotide sequence. Three different RNA aptamers were obtained after 11 rounds of SELEX using isolated rat adult CMs. The amounts of these aptamers taken up by CMs were significantly higher than those by cells other than CM. To examine whether these aptamers can be used as an intracellular DDS, we conjugated them to the phospholamban RNA aptamer. We previously reported that the latter aptamer binds phospholamban and enhances the Ca2+ transport activity of the sarcoplasmic reticulum (SR) within CM. The conjugated aptamers were indeed taken up by CMs and enhanced the Ca2+ transport activity of SR, facilitating contraction and relaxation of CMs. The present study thus provides a novel tool for the heart-specific intracellular DDS.

Generation of HBsAg DNA aptamer using modified cell-based SELEX strategy.

Aptamers as potential alternatives for antibodies could be employed against hepatitis B surface antigen (HBsAg), the great hallmark and first serological marker in HBV, for further theragnostic applications. Therefore, isolation HBsAg specific aptamer was performed in this study with a modified Cell-SELEX method. HEK293T overexpressing HBsAg and HEK293T as target and control cells respectively, were incubated with single-stranded rounds of DNA library during six SELEX and Counter SELEX rounds. Here, we introduced the new modified Cell-SELEX using deoxyribonuclease I digestion to separate single stranded DNA aptamers against the HBsAg. Characterization and evaluation of selected sequences were performed using flow cytometry analysis. The results led to isolation of 15 different ssDNA clones in six rounds of selection which were categorized to four clusters based on common structural motifs. The evaluation of SELEX progress showed growth in aptamer affinity with increasing in the cycle number. Taken together, the application of modified cell-SELEX demonstrated the isolation of HBsAg-specific ssDNA aptamers with proper affinity. Modified cell-SELEX as an efficient method can shorten the selection procedure and increase the success rate while the benefits of cell-based SELEX will be retained. Selected aptamers could be applied in purification columns, diagnostic kits, and drug delivery system against HBV-related liver cancer.

Rationally Designed Multivalent Aptamers Targeting Cell Surface for Biomedical Applications.

Specific interactions between ligands and receptors on cell surface play an important role in the cell biological process. Nucleic acid aptamers as commonly used ligands enable specific recognition and tight binding to membrane protein receptors for modulation of cell fate. Therefore, molecular probes with aptamers can be applied for cancer diagnosis and targeted therapy by targeting overexpression membrane proteins of cancer cells. However, because of their fast degradation and rapid glomerulus clearance in vivo, the applications of aptamers in physiological conditions remain challenged. Inspired by natural multivalent interactions, many approaches have been developed to construct multivalent aptamers to improve the performance of aptamers in complex matrices with higher binding affinity, more stability, and longer circulation time. In this review, we first introduce the aptamer generation from purified protein-based SELEX and whole cell-based SELEX for targeting the cell surface. We then highlight the approaches to fabricate multivalent aptamers and discuss their properties. By integrating different materials (including inorganic nanomaterials, diacyllipid, polymeric nanoparticles, and DNA nanostructures) as scaffolds with an interface modification technique, we have summarized four kinds of multivalent aptamers. After that, representative applications in biosensing and targeted therapy are illustrated to show the elevated performance of multivalent aptamers. In addition, we analyze the challenges and opportunities for the clinical practices of multivalent aptamers.

Aptamers and Antisense Oligonucleotides for Diagnosis and Treatment of Hematological Diseases.

Aptamers or chemical antibodies are single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies. Aptamers can be easily produced in vitro through a process known as systemic evolution of ligands by exponential enrichment (SELEX) or a cell-based SELEX procedure. Aptamers and modified aptamers, such as slow, off-rate, modified aptamers (SOMAmers), can bind to target molecules with less polar and more hydrophobic interactions showing slower dissociation rates, higher stability, and resistance to nuclease degradation. Aptamers and SOMAmers are largely employed for multiplex high-throughput proteomics analysis with high reproducibility and reliability, for tumor cell detection by flow cytometry or microscopy for research and clinical purposes. In addition, aptamers are increasingly used for novel drug delivery systems specifically targeting tumor cells, and as new anticancer molecules. In this review, we summarize current preclinical and clinical applications of aptamers in malignant and non-malignant hematological diseases.

A Novel Aptamer LL4A Specifically Targets Vemurafenib-Resistant Melanoma through Binding to the CD63 Protein

Melanoma is a highly aggressive tumor with a poor prognosis, and half of all melanoma patients harbor BRAF mutations. A BRAF inhibitor, vemurafenib (PLX4032), has been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) to treat advanced melanoma patients with BRAFV600E mutation. However, the efficacy of vemurafenib is impeded by adaptive resistance in almost all patients. In this study, using a cell-based SELEX (systematic evolution of ligands by exponential enrichment) strategy, we obtained a DNA aptamer (named LL4) with high affinity and specificity against vemurafenib-resistant melanoma cells. Optimized truncated form (LL4A) specifically binds to vemurafenib-resistant melanoma cells with dissociation constants in the nanomolar range and with excellent stability and low toxicity. Meanwhile, fluorescence imaging confirmed that LL4A significantly accumulated in tumors formed by vemurafenib-resistant melanoma cells, but not in control tumors formed by their corresponding parental cells in vivo. Further, a transmembrane protein CD63 was identified as the binding target of aptamer LL4A using a pull-down assay combined with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. CD63 formed a supramolecular complex with TIMP1 and β1-integrin, activated the nuclear factor кB (NF-кB) and mitogen-activated protein kinase (MAPK) signaling pathways, and contributed to vemurafenib resistance. Potentially, the aptamer LL4A may be used diagnostically and therapeutically in humans to treat targeted vemurafenib-resistant melanoma.

Selection and characterization of novel DNA aptamer against colorectal carcinoma Caco-2 cells.

Aptamers are short, single-stranded nucleic acid (DNA or RNA) oligonucleotides that can be obtained by a technique called systematic evolution of ligands by exponential enrichment (SELEX) in vitro. Due to superior properties such as small size, high binding affinity, and stability, they are considered to be feasible tools for diagnosis and treatment of disease. In the current study, we attempted to screen a high-affinity DNA aptamer to selectively target the colorectal carcinoma Caco-2 cells by using cell-based SELEX approach. After 14 consecutive rounds of selection, aptamer ApC1 was identified. Confocal microscopy results revealed that ApC1 could rapidly internalize into Caco-2 cells but not HEK 293 cells. Moreover, it showed high specificity to Caco-2 cells rather than other cell lines such as 293T, HeLa, MCF-7, HL-60, and NB4. Collectively, our results demonstrated that aptamer ApC1 has high specificity to colorectal carcinoma Caco-2 cells, which could be further applied for targeted therapy of colorectal cancer in future studies.

A CTLA-4 Antagonizing DNA Aptamer with Antitumor Effect.

The successful translation of cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade has revolutionized the concept of cancer immunotherapy. Although monoclonal antibody therapeutics remain the mainstream in clinical practice, aptamers are synthetic oligonucleotides that encompass antibody-mimicking functions. Here, we report a novel high-affinity CTLA-4-antagonizing DNA aptamer (dissociation constant, 11.84 nM), aptCTLA-4, which was identified by cell-based SELEX and high-throughput sequencing. aptCTLA-4 is relatively stable in serum, promotes lymphocyte proliferation, and inhibits tumor growth in cell and animal models. Our study demonstrates the developmental pipeline of a functional CTLA-4-targeting aptamer and suggests a translational potential for aptCTLA-4.

Abstract 1606: Targeting human T regulatory cells with novel Interleukin 2 alpha - IL2 complex-specific RNA aptamer

Abstract Background: RNA aptamers are small RNA molecules that bind antigens like antibodies and are currently being explored as alternatives to antibodies for diagnosis and therapy. A potential merit of aptamers is that they can be generated against native cellular antigens, such as those with unique post-translational modifications or receptor-ligand complexes, for which antibody generation can be difficult. Here, we report the use of a cell-based systematic enrichment approach (SELEX) to develop a novel Treg-binding RNA aptamer specific to IL2Rα-IL2 receptor-ligand complex. Methods and Results: A. Generation of Treg-binding aptamers: We designed a cell-based SELEX strategy to generate RNA aptamers specific to human T regulatory (Treg) cells. The starting library consisted of random RNA aptamers with a structural diversity of ~1012. Aptamers against common T cell antigens were pre-cleared using CD4+CD25- Teff cells. Treg-binding aptamers were then positively selected using CD4+CD25+ Tregs from the same donor. After amplification of Treg binders by RT-PCR, the whole selection was repeated eight times, each time with T cells from a different donor. At the end, SELEX-enriched aptamer pools predominantly bound to Tregs, but not to Teff cells, which were then sequenced. The most prevalent Treg-binder, Tr-1, showed 63,875-fold enrichment by the end of SELEX (from 0.36 copies in the starting library to 22,995 copies in the eighth round per million total reads). B. Tr-1 binds to IL2Rα- IL2 complex We tested if Tr-1 recognized human IL2Rα by measuring Tr-1 binding to recombinant IL2Rα protein using RT-qPCR. Results indicated that Tr-1 bound to IL2Rα and showed ~3-fold higher binding to IL2Rα when IL2 was added indicating that Tr-1 recognizes either the receptor-ligand complex or a conformational change in IL2-bound IL2Rα. Binding of Tr-1 to IL2Rα did not significantly alter its affinity to IL2. C. Tr-1 inhibits Treg induction Transformed B cells can induce Treg development in the tumor microenvironment. Experimentally, this was evaluated by quantifying Tregs generated from autologous CD4+ T cells co-cultured with Epstein-Barr virus-transformed B (EBV-B) cells. Addition of Tr-1 resulted in ~30% reduction in EBV-B-induced Tregs (p=0.017). Conclusion: We used a Treg cell-based SELEX strategy to derive a novel Treg-binding RNA aptamer (Tr-1) that preferentially binds to the IL2Rα-IL2 complex. Tr-1 reduced Treg induction by transformed B cells suggesting its potential as a therapeutic agent to reduce tumor-induced immunosuppression. Ongoing studies are further exploring its use in Treg inhibition and in targeting receptor-ligand complexes in cancer. While aptamers recognizing cellular receptors exist, to our knowledge, this is the first report of an aptamer recognizing a receptor-ligand complex. Our approach to generating aptamers against receptor-ligand complexes could have huge scientific impact. Citation Format: Suresh Veeramani, Sue E. Blackwell, William H. Thiel, Paloma H. Giangrande, George J. Weiner. Targeting human T regulatory cells with novel Interleukin 2 alpha - IL2 complex-specific RNA aptamer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2017-1606

Selection of Nucleic Acid Aptamers Targeting Tumor Cell-Surface Protein Biomarkers.

Aptamers are nucleic acids referred to as chemical antibodies as they bind to their specific targets with high affinity and selectivity. They are selected via an iterative process known as ‘selective evolution of ligands by exponential enrichment’ (SELEX). Aptamers have been developed against numerous cancer targets and among them, many tumor cell-membrane protein biomarkers. The identification of aptamers targeting cell-surface proteins has mainly been performed by two different strategies: protein- and cell-based SELEX, when the targets used for selection were proteins and cells, respectively. This review aims to update the literature on aptamers targeting tumor cell surface protein biomarkers, highlighting potentials, pitfalls of protein- and cell-based selection processes and applications of such selected molecules. Aptamers as promising agents for diagnosis and therapeutic approaches in oncology are documented, as well as aptamers in clinical development.

Selective identification of specific aptamers for the detection of non-typhoidal salmonellosis in an apta-impedimetric sensing format

The authors report on an aptamer-based method for the detection of S. Typhimurium. The aptamers were identified by using a modified cell-based SELEX method (cell-SELEX) by adopting an alternating negative-positive selection strategy. Each cross-reactive population was used separately, with a focus on generating strain-specific aptamers in order to enhance selectivity. The aptamers were characterized and chemically modified with thiol groups at the 5′ end which enables covalent binding to gold nanoparticles on a screen printed carbon electrode (GNP-SPCE). The aptamer-modified GNP-SPCE was subsequently applied to impedimetric sensing of S. Typhimurium in water and food samples. Response is linear in the 10 to 105 CFU⋅mL−1 concentration range, and the limit of detection is ~10 CFU⋅mL−1. The assay can distinguish S. Typhimurium from S. Typhi and has been validated in water and spiked egg samples.

P2.03b-090 A CTLA-4 Antagonizing DNA Aptamer with Anti-Tumor Effect: Topic: Biomarkers

The discovery of cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade and its successful clinical translation has revolutionized the concept of cancer immunotherapy. Although immunecheckpoint-targeting antibody has shown impressive results in a diverse array of cancers, their cell-based manufacturing process influences production capacity and cause variation between batches. Aptamers are synthetic DNA or RNA oligonucleotides that encompass antibody-mimicking functions. With its chemically synthetic nature, aptamer can be produced in large scale with controllable batch variations and lower manufacturing cost. Here, we report the development of a CTLA-4 antagonizing DNA aptamer, termed aptCTLA-4, using the cell-based SELEX and next-generation sequencing. The aptCTLA-4 exhibits good binding affinity (dissociation constant, 11.8 nM). In vitro lymphocyte proliferation assays demonstrated that the aptCTLA-4 promotes T cell proliferation, and in vivo murine syngeneic tumor models further revealed its tumor-inhibitory effects. Our data suggest the translational potential of the aptCTLA-4 to be developed into a therapeutic aptamer.

Developing Aptamers by Cell-Based SELEX.

The reliable targeting of cell surface disease-associated proteins is a major challenge in chemical biology and molecular medicine. In this regard, aptamers represent a very attractive and innovative class of ligand molecules. Aptamers are generated by a reiterated in vitro procedure, named SELEX (Systematic Evolution of Ligands by Exponential enrichment). In order to generate aptamers for heavily modified cell surface-bound proteins and transmembrane receptors, the SELEX procedure has been recently adapted to the use of living cells as complex targets (referred as "cell-SELEX"). Here we give an overview on the most recent advances in the field of cell-SELEX technology, providing a detailed description of the differential cell-SELEX approach that has been developed in our laboratory to identify specific signatures for human malignant glioma and non-small-cell lung cancer. The procedures used for the evaluation of binding specificity and for the preliminary identification of potential target receptors will be also described.

Generating Cell Targeting Aptamers for Nanotheranostics Using Cell-SELEX.

Detecting and understanding changes in cell conditions on the molecular level is of great importance for the accurate diagnosis and timely therapy of diseases. Cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), a foundational technology used to generate highly-specific, cell-targeting aptamers, has been increasingly employed in studies of molecular medicine, including biomarker discovery and early diagnosis/targeting therapy of cancer. In this review, we begin with a mechanical description of the cell-SELEX process, covering aptamer selection, identification and identification, and aptamer characterization; following this introduction is a comprehensive discussion of the potential for aptamers as targeting moieties in the construction of various nanotheranostics. Challenges and prospects for cell-SELEX and aptamer-based nanotheranostic are also discussed.

Targeting Insulin Receptor with a Novel Internalizing Aptamer

Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

Evolution of Cell-Type-Specific RNA Aptamers Via Live Cell-Based SELEX.

Live cell-based SELEX (Systematic Evolution of Ligand EXponential enrichment) is a promising approach for identifying aptamers that can selectively bind to a cell-surface antigen or a particular target cell population. In particular, it offers a facile selection strategy for some special cell-surface proteins that are original glycosylated or heavily post-translationally modified, and are unavailable in their native/active conformation after in vitro expression and purification. In this chapter, we describe evolution of cell-type-specific RNA aptamers targeting the human CCR5 by combining the live cell-based SELEX strategy with high-throughput sequencing (HTS) and bioinformatics analysis.

Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX

Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

142. Evolution of Cell-Specific RNA Aptamers Against Glioblastoma Cancer Stem Cells Via Live Cell-Based SELEX

Glioblastoma (GBM) is the most prevalent and lethal primary brain tumour and ranks among the most lethal of all cancers. The median survival time is only 12 to 15 months despite surgical resection followed by radio- and chemotherapy. Cancer stem cells (CSCs), also called self-renewing tumour-initiating cells (TICs), represent a malignant subpopulation of cells in hierarchically organized tumours. GBM CSCs are thought to be portion of glioblastoma cells that are responsible for both cellular invasion and therapeutics resistance, indicating the need for therapies that kill these cells and for imaging reagents that can identify them in vivo. Although several surface CSCs markers have shown their promise in glioblastoma to prospectively enrich or deplete CSCs, their uses were limited by a shared antigen with normal stem cells. Novel biomarkers that specifically recognize CSCs are highly desired. Therefore, developing a cell-type specific aptamers that prospectively identify GBM CSCs may not only serve as a platform for the development of novel targeted therapies and imaging reagents, but also permit the discovery of novel biomarkers for GBM CSCs. Additionally, we reasoned that a combinatorial use of various nucleic acids such as small interfering RNAs (siRNAs) and aptamers could be more efficacious in suppressing tumour growth through specifically targeting to GBM CSCs.In the present study, by combining the “Live Cell-based SELEX” strategy with high throughput sequencing (HTS) technology and bioinformatics analysis, we successfully identified several 2’-Fluoropyrimidine modified RNA aptamers targeted to Glioblastoma cancer stem cells. One of the best candidates (S-2 aptamer) specifically bound and was efficiently internalized into Glioblastoma cancer stem cells. Our efforts demonstrate that the cell-specific, internalizing RNA aptamers offer great promise for cell-type- or tissue-specific delivery and imaging reagents for targeted treatment of glioblastoma. We are presently investigating the ligand recognized by the aptamer.