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. 2015 Jun;25(3):162-72.
doi: 10.1089/nat.2014.0522. Epub 2015 Apr 20.

Identification of epithelial ovarian tumor-specific aptamers

Gregory Benedetto  1 , Timothy J Hamp  1   2 , Peter J Wesselman  1 , Christine Richardson  1
Affiliations

Identification of epithelial ovarian tumor-specific aptamers

Gregory Benedetto et al. Nucleic Acid Ther. 2015 Jun.

Abstract

Ovarian cancer is often diagnosed in late stages with few treatment options and poor long-term prognosis. New clinical tools for early detection of ovarian malignancies will significantly help reduce mortality and improve current long-term survival rates. The objective of this work was to identify ovarian tumor-specific single-stranded DNA aptamers that bind to malignant ovarian tumor cells and internalize with high affinity and specificity. Aptamers can identify unique tumor biomarkers, can aid in early detection and diagnosis of neoplastic disorders, and can be functionalized by conjugation to small molecules. To identify aptamers from random single-stranded DNA pools (60 bases long), we used whole Cell-SELEX (systematic evolution of ligands by exponential enrichment) to enrich and isolate tumor-specific aptamers that bind to tumor-specific receptors in their native state on the cell surface. Next-Generation sequencing identified seven novel aptamers and detailed analyses of three are described. Aptamers bound to, and were internalized by, target Caov-3 cell populations, but not nontarget nonmalignant ovarian epithelial HOSE 6-3 cells or multiple other epithelial tumor cell lines. Furthermore, aptamers showed unique binding affinities with apparent dissociation constants (Kd) measuring in the submicromolar range supporting their physiological relevance and potential use in clinical applications.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Monitoring the enrichment of Caov-3-specific aptamers from rounds 3, 8, and 12 of Cell-SELEX by flow cytometry. Flow cytometry histograms are shown for Cy5-labeled aptamer pools from successive rounds of Cell-SELEX. A right shift in fluorescence of the cell populations is indicative of increases in aptamer binding and internalization. Aptamer rounds of selection: initial random library (formula image), round 3 (formula image), round 8 (formula image), and round 12 (formula image) of Cell-SELEX.
<b>FIG. 2.</b>
FIG. 2.
Binding kinetics of ovarian tumor-specific aptamers to determine equilibrium dissociation constants (Kd) to ovarian tumor cell lines. To assess target cell and aptamer binding affinities, we used flow cytometry to quantify Cy5 aptamer conjugate binding and specificity to ovarian epithelial malignant cell lines Caov-3 (●), SK-OV-3 (■), and SW626 (▲). Data points represent the average fluorescent events observed (n=3, error bars±SD) at indicated nM concentrations. (A) Cy5-RLA01 aptamer conjugates with Caov-3 target cells and nontarget SK-OV-3 and SW626 cell lines in increasing nM doses. (B) Cy5-RLA02 aptamer conjugates with Caov-3 target cells and nontarget SK-OV-3 and SW626 cell lines in increasing nM doses. (C) Cy5-RLA03 aptamer conjugates with Caov-3 target cells and nontarget SK-OV-3 and SW626 cell lines in increasing nM doses. Individual apparent Kd values were calculated by using the equation Y=Bmax*X^h/(Kd^h+X^h) for the three DNA aptamers.
<b>FIG. 3.</b>
FIG. 3.
Dose-dependent and time-dependent specificity of RLA01, RLA02, and RLA03 aptamers binding to target cells. RLA01 aptamers were incubated at increasing concentrations and times to normal and malignant epithelial cell lines. (A) Flow cytometry analysis of Cy5-RLA01, RLA02, and RLA03 incubated with indicated cell lines for 2 h. Aptamer doses corresponding to colored histograms are control (formula image), 400 (formula image), and 800 nM (formula image) concentrations. (B) Cy5-RLA01 incubated with indicated cell lines for 4 h. (C) Confocal imaging of indicated cell lines treated with Cy5-RLA01 imaged at 60×using a nuclear stain (DAPI as blue), a membrane stain (WGA-Alexa Fluor 488 as green), and Cy5 aptamers (Cy5 pseudocolored as yellow). (D) Binding kinetics of RLA01 when increasing molar concentrations are incubated with Caov-3 (•), SK-OV-3 (▪), and HeLa (▴). Data points represent the average fluorescent events observed (n=3, error bars±SD) at indicated nM concentrations, and individual apparent Kd values were calculated by using the equation Y=Bmax*X^h/(Kd^h+X^h).
<b>FIG. 4.</b>
FIG. 4.
Internalization of RLA01, RLA02, and RLA03 aptamers into Caov-3 cells occurs through the endocytic pathway. (A) Caov-3 cells were treated with Cy5-conjugated RLA01, RLA02, or RLA03 aptamers. Untreated (-) and Cy5-conjugated RLA01, RLA02, and RLA03 aptamer-treated cells were imaged at 60×using a nuclear stain (DAPI as blue), a membrane stain (WGA-Alexa Fluor 488 as green), and Cy5 aptamers (Cy5 pseudocolored as yellow). (B) Graphic representation of Cy5-RLA01 (500 nM) fluorescent events by flow cytometry observed over a 2-h time course. (C) Graphic representation of percentage of cells showing positive endocytic internalization of Cy5-RLA01 (500 nM) confirmed using pHrodo Red Transferrin Conjugate. (D) Caov-3 cells were treated with 5 μM Cy5-conjugated RLA01, RLA02, or RLA03 aptamers. Cells were imaged at 60× using a nuclear stain (DAPI as blue), an endosomal-specific marker (pHrodo Red pseudocolored as red), and Cy5 aptamers (Cy5 pseudocolored as yellow).
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