Identification of cell membrane protein stress-induced phosphoprotein 1 as a potential ovarian cancer biomarker using aptamers selected by cell systematic evolution of ligands by exponential enrichment.

In this paper, we describe the elucidation of the target of an aptamer against ovarian cancer previously obtained by cell-SELEX (SELEX = systematic evolution of ligands by exponential enrichment). The target's identity, stress-induced phosphoprotein 1 (STIP1), was determined by mass spectrometry and validated by flow cytometry, using siRNA silencing and protein blotting. Initial oncologic studies show that the aptamer inhibits cell invasion, indicating that STIP1, which is currently under investigation as a potential biomarker for ovarian cancer, plays a critical role in this process. These results serve as an excellent example of how protein target identification of aptamers obtained by cell-SELEX can serve as a means to identify promising biomarker candidates and can promote the development of aptamers as a new drug class to block important oncological processes.

RNA aptamers specific for transmembrane p24 trafficking protein 6 and Clusterin for the targeted delivery of imaging reagents and RNA therapeutics to human ß cells.

The ability to detect and target ß cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human ß cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human ß cells. The putative targets of the two aptamers are transmembrane p24 trafficking protein 6 (TMED6) and clusterin (CLUS). When given systemically in immune deficient mice, these aptamers recognize the human islet graft producing a fluorescent signal proportional to the number of human islets transplanted. These aptamers cross-react with endogenous mouse ß cells and allow monitoring the rejection of mouse islet allografts. Finally, once conjugated to saRNA specific for X-linked inhibitor of apoptosis (XIAP), they can efficiently transfect non-dissociated human islets, prevent early graft loss, and improve the efficacy of human islet transplantation in immunodeficient in mice.

Aptamers against mouse and human tumor-infiltrating myeloid cells as reagents for targeted chemotherapy.

Local delivery of anticancer agents has the potential to maximize treatment efficacy and minimize the acute and long-term systemic toxicities. Here, we used unsupervised systematic evolution of ligands by exponential enrichment to identify four RNA aptamers that specifically recognized mouse and human myeloid cells infiltrating tumors but not their peripheral or circulating counterparts in multiple mouse models and from patients with head and neck squamous cell carcinoma (HNSCC). The use of these aptamers conjugated to doxorubicin enhanced the accumulation and bystander release of the chemotherapeutic drug in both primary and metastatic tumor sites in breast and fibrosarcoma mouse models. In the 4T1 mammary carcinoma model, these doxorubicin-conjugated aptamers outperformed Doxil, the first clinically approved highly optimized nanoparticle for targeted chemotherapy, promoting tumor regression after just three administrations with no detected changes in weight loss or blood chemistry. These RNA aptamers recognized tumor infiltrating myeloid cells in a variety of mouse tumors in vivo and from human HNSCC ex vivo. This work suggests the use of RNA aptamers for the detection of myeloid-derived suppressor cells in humans and for a targeted delivery of chemotherapy to the tumor microenvironment in multiple malignancies.

Nucleic acid aptamers for biosensors and bio-analytical applications.

Oligonucleotides were once considered only functional as molecules for the storage of genetic information. However, the discovery of RNAzymes, and later, DNAzymes, unravelled the innate potential of oligonucleotides in many other biological applications. In the last two decades, these applications have been further expanded through the introduction of Systematic Evolution of Ligands by EXponential enrichment (SELEX) which has generated, by repeated rounds of in vitro selection, a type of molecular probe termed aptamers. Aptamers are oligonucleic acid (or peptide) molecules that can bind to various molecular targets and are viewed as complements to antibodies. Aptamers have found applications in many areas, such as bio-technology, medicine, pharmacology, microbiology, and analytical chemistry, including chromatographic separation and biosensors. In this review, we focus on the use of aptamers in the development of biosensors. Coupled with their ability to bind a variety of targets, the robust nature of oligonucleotides, in terms of synthesis, storage, and wide range of temperature stability and chemical manipulation, makes them highly suitable for biosensor design and engineering. Among the many design strategies, we discuss three general paradigms that have appeared most frequently in the literature: structure-switching, enzyme-based, and aptazyme-based designs.

Molecular recognition of live methicillin-resistant staphylococcus aureus cells using DNA aptamers.

AIM: To generate DNA-aptamers binding to Methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The Cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology was used to run the selection against MRSA bacteria and develop target-specific aptamers. MRSA bacteria were targeted while Enterococcus faecalis bacteria were used for counter selection during that process. Binding assays to determine the right aptamer candidates as well as binding assays on clinical samples were performed through flow cytometry and analyzed using the FlowJo software. The characterization of the aptamers was done by determination of their Kd values and determined by analysis of flow data at different aptamer concentration using SigmaPlot. Finally, the recognition of the complex Gold-nanoparticle-aptamer to the bacteria cells was observed using transmission electron microscopy (TEM). RESULTS: During the cell-SELEX selection process, 17 rounds were necessary to generate enrichment of the pool. While the selection was run using fixed cells, it was shown that the binding of the pools with live cells was giving similar results. After sequencing and analysis of the two last pools, four sequences were identified to be aptamer candidates. The characterization of those aptamers showed that based on their Kd values, DTMRSA4 presented the best binding with a Kd value of 94.61 ± 18.82 nmol/L. A total of ten clinical samples of MRSA , S. aureus and Enterococcus faecalis were obtained to test those aptamers and determine their binding on a panel of samples. DTMRSA1 and DTMRSA3 showed the best results regarding their specificity to MRSA , DTMRSA1 being the most specific of all. Finally, those aptamers were coupled with gold-nanoparticle and their binding to MRSA cells was visualized through TEM showing that adduction of nanoparticles on the aptamers did not change their binding property. CONCLUSION: A total of four aptamers that bind to MRSA were obtained with Kd values ranking from 94 to 200 nmol/L.

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 K(d')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.

Selection of aptamers specific for adipose tissue.

BACKGROUND: Obesity has reached epidemic proportions, affecting more than one tenth of the world's population. As such, adipose tissue is being increasingly recognized as an important therapeutic target for obesity and related metabolic disorders. While many potential targets of adipose tissue have been established and drugs developed, very few of those drugs specifically target adipose tissue without affecting other tissue. This results from a limited knowledge of both cell-surface markers and physicochemical traits specific to adipocytes that might otherwise be exploited by circulating drugs. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the use of cell-SELEX technology to select two aptamers that can specifically recognize mature adipocytes: adipo-1 and adipo-8. Adipo-8 shows high affinity for differentiated, mature 3T3-L1 adipocytes with a K(d) value of 17.8±5.1 nM. The binding was sustained upon incubation at 37°C and insulin stimulation, but was lost upon trypsin treatment. The binding ability was also verified on frozen tissue slides with low background fluorescence and isolated adipocytes. CONCLUSIONS/SIGNIFICANCE: Aptamer adipo-8 selected from a random library appears to bind to mature differentiated adipocytes specifically. This aptamer holds great promise as a molecular recognition tool for adipocyte biomarker discovery or for targeted delivery of molecules to adipocytes.

Study of the molecular recognition of aptamers selected through ovarian cancer cell-SELEX.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment. METHODOLOGY/PRINCIPAL FINDINGS: Cell-SELEX technology was employed to develop new molecular probes for ovarian cancer cell surface markers. A total of thirteen aptamers with K(d)'s to ovarian cancer cells in the pico- to nanomolar range were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also performed. CONCLUSIONS/SIGNIFICANCE: We have selected a series of aptamers that bind to different types of ovarian cancer, but not cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of biomarkers that are related to cancer.