Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX

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.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.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

Selection of Aptamers Specific for Adipose Tissue

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.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.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

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 K(d) 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 K(d) values, DTMRSA4 presented the best binding with a K(d) 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 K(d) values ranking from 94 to 200 nmol/L

Abstract PR16: RNA aptamers specific for tumor-infiltrating myeloid cells

Abstract Myeloid cells have been extensively studied in the tumor microenvironment for their role in promoting cancer cell survival and metastases. However, their peculiar activated phenotype has not been exploited for the targeted delivery of anticancer agents to the tumor site. Here we report a new therapeutic strategy that allows the delivery of cancer drugs to both tumor and metastases by the preferential targeting of genetically stable tumor-infiltrating myeloid cells (TIMC). We identified 4 monoclonal RNA aptamers that specifically recognize myeloid-derived suppressor cells (MDSC) and macrophages infiltrating the tumor, but not the splenic counterparts. The use of these aptamers conjugated to doxorubicin greatly enhances the accumulation of the chemotherapeutic drug in the primary and metastatic tumor sites in multiple cancer models. Compared to their individual components or current therapeutic approaches, doxorubicin-conjugated TIMC-specific aptamers show enhanced anticancer efficacy and no detectable treatment-related toxicity. This strategy has the intrinsic potential to target multiple tumor types through the same reagents. This abstract is also being presented as Poster A35. Citation Format: Adriana De La Fuente, Jimmy Caroli, Dimitri Van Simaeys, Serena Zilio, Emilia Mazza, Vincenzo Bronte, Silvio Bicciato, Paolo Serafini. RNA aptamers specific for tumor-infiltrating myeloid cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR16

Confocal images of aptamers stained with cultured differentiated 3T3-L1 adipocytes.

<p>Cells were incubated with aptamers conjugated with biotin, and binding events were observed with PE-conjugated streptavidin. (A) unselected PE-labeled library; (B) adipo-1; (C) and adipo-8. The final concentration of the aptamers in the binding buffer was 250 nM. <i>(Left</i>) fluorescence and (<i>Right</i>) optical images of differentiated 3T3-L1 adipocytes.</p

Preliminary determination of the nature of the molecular targets to which aptamers bind.

<p>Effect of trypsin treatment on the binding ability of adipo-1 (A) and adipo-8 (B) to differentiated 3T3-L1 adipocytes. The concentration of aptamers in the binding buffer was 250 nM.</p

Flow cytometry assays to assess if aptamers’ binding ability to mature adipocytes can be influenced by temperature and insulin stimulation.

<p>Effects of temperature (A, B) and insulin stimulation (C) on the binding ability of adipo-1 and adipo-8 (250 nM) to differentiated 3T3-L1 adipocytes.</p

Molecular recognition of adipo-8 with isolated adipocyte.

<p>Fluorescence image of isolated mature adipocyte stained by cy5-labled adipo-8 (A), library (B). Fluorescence image of isolated pelleted cells of from collagenase-digested adipose tissue stained by cy5-labled adipo-8 (C). The final concentration of the aptamers in the binding buffer was 250 nM. Optical (Left) and fluorescence (Right) image of adipocyte and pelleted cells.</p