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

Title:
Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing (Abstract POSTER-THER-1429)
Authors:
Whelan, Rebecca J. ( 0000-0002-9293-1528 ) ; Kapur, Arvinder; Felder, Mildred; Shallcross, Jamie A.; Nie, Jeff; Patankar, Manish S.
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.
Citation:
Whelan, Rebecca J., Arvinder Kapur, Mildred Felder, Jamie Shallcross, Jeff Nie, and Manish S. Patankar. 2015. "Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing (Abstract POSTER-THER-1429)." Clinical Cancer Research 21: Supplement 16.
Publisher:
American Association for Cancer Research
DATE ISSUED:
2015-08-13
Department:
Chemistry and Biochemistry
Type:
Abstract
PUBLISHED VERSION:
10.1158/1557-3265.OVCASYMP14-POSTER-THER-1429
Additional Links:
http://clincancerres.aacrjournals.org/lookup/doi/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.
PERMANENT LINK:
http://hdl.handle.net/11282/594048

Full metadata record

DC FieldValue Language
dc.contributor.authorWhelan, Rebecca J.en
dc.contributor.authorKapur, Arvinderen
dc.contributor.authorFelder, Mildreden
dc.contributor.authorShallcross, Jamie A.en
dc.contributor.authorNie, Jeffen
dc.contributor.authorPatankar, Manish S.en
dc.date.accessioned2016-01-19T13:30:33Zen
dc.date.available2016-01-19T13:30:33Zen
dc.date.issued2015-08-13en
dc.identifier.citationWhelan, Rebecca J., Arvinder Kapur, Mildred Felder, Jamie Shallcross, Jeff Nie, and Manish S. Patankar. 2015. "Selection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing (Abstract POSTER-THER-1429)." Clinical Cancer Research 21: Supplement 16.en
dc.identifier.issn1078-0432en
dc.identifier.urihttp://hdl.handle.net/11282/594048en
dc.description.abstractHumanized 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.en_US
dc.language.isoen_USen
dc.publisherAmerican Association for Cancer Researchen
dc.identifier.doi10.1158/1557-3265.OVCASYMP14-POSTER-THER-1429en
dc.relation.urlhttp://clincancerres.aacrjournals.org/lookup/doi/10.1158/1557-3265.OVCASYMP14-POSTER-THER-1429en
dc.subject.departmentChemistry and Biochemistryen_US
dc.titleSelection of DNA aptamers for an ovarian cancer cell line using high-throughput sequencing (Abstract POSTER-THER-1429)en_US
dc.typeAbstracten
dc.description.notesIn Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR.en_US
dc.identifier.journalClinical Cancer Researchen
dc.identifier.volume21en_US
dc.rightsArchived with thanks to Clinical Cancer Researchen
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