Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing (Abstract POSTER-THER-1429)

Abstract

Humanized antibodies have been extensively investigated as therapeutic as well as diagnostic agents. While the antigen specificity offered by antibodies makes them very attractive for such theranostic applications, their large-scale synthesis can be challenging and expensive. We are therefore investigating alternate strategies to develop agents that can be used for in vivo monitoring as well as for treatment of epithelial ovarian tumors. One approach is to develop Single Stranded DNA aptamers that selectively bind to ovarian cancer cells. The ssDNA aptamers can be synthesized using template-driven or de novo chemical synthetic approaches to manufacture agents at a large scale and low cost. The challenge however, is to develop aptamers that are specific to ovarian cancer cells. In the current study, we report a streamlined approach that incorporates the cell-based Systematic Evolution of Ligands by Exponential Enrichment (cell-SELEX) with DNASeq technology to select aptamers that recognize ovarian cancer cells. An ssDNA aptamer library composed of ~1015 sequences was subjected to ten iterative rounds of selection against the ovarian cancer cell line OVCAR-3. Aptamers from each round were amplified by asymmetric PCR and subjected to high-throughput sequencing. Eight ssDNA aptamers enriched through the selection process were identified by DNASeq and subsequent bioinformatics analysis and their selectivity and affinity for OVCAR-3 cells was determined by flow cytometry. Two of these aptamers (Apt-1 and Apt-8) showed significant binding to OVCAR-3 cells with Kd of 24 and 28 nM, respectively. Secondary structure analysis using mfold indicated that Apt-1 and Apt-8 had defined secondary structures resulting from ordered base pairing of the ssDNA. The inclusion of high-throughput sequencing techniques has therefore allowed rapid identification of theranostic aptamers from an large randomized library of ssDNA sequences. Our ongoing experiments are focused on coupling of the ovarian cancer cell-specific ssDNA aptamers to contrast agents or cytotoxic drugs. The ssDNA aptamers coupled to contrast agents are specifically being investigated for in vivo imaging of ovarian cancer masses in the peritoneum whereas the aptamers coupled to drugs can be used for the treatment of ovarian cancer.

Publisher

American Association for Cancer Research

Publication Date

8-13-2015

Publication Title

Clinical Cancer Research

Department

Chemistry and Biochemistry

Document Type

Abstract

DOI

https://dx.doi.org/10.1158/1557-3265.OVCASYMP14-POSTER-THER-1429

Notes

In Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR.

Language

English

Format

text

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