Use of Cell-SELEX to Generate DNA Aptamers as Molecular Probes of HPV-Associated Cervical Cancer Cells

Jessica C Graham,Helmut Zarbl,Eric Deutsch

doi:10.1371/journal.pone.0036103

Abstract

BackgroundDisease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the approach used to select high affinity aptamers for specific macromolecular targets from among the >1013 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines.Methodology/Principal FindingsWhole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX)). Using this approach, we identified high affinity aptamers (nanomolar range Kd) to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa). We also performed preliminary investigation of the aptamer epitopes and their binding characteristics.Conclusions/SignificanceUsing AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of cancer biopsies and aid in the effective management of pathological conditions (diagnosis, predicted outcome, and treatment options).

Highlights

Cervical cancer is the second most common cancer affecting women worldwide [1]
Aptamers were targeted to the nontumorigenic HF cell line, using isogenic HeLa cells for negative counter-selection
After eighteen cycles of positive/ negative selection we identified molecular probes that were highly specific for the revertant HF cell line (Table 1)

Summary

Introduction

Cervical cancer is the second most common cancer affecting women worldwide [1]. More than 90% of cervical cancers are caused by HPV, and approximately 10,800 new cases of HPVrelated cervical cancer are diagnosed in the United States (US) annually [2]. HPV type 16 (HPV-16) is associated with more than 50% of cervical cancers worldwide [3]. Despite the availability of a preventive vaccine for HPV infection [5], there remains a significant need for the development of biomarkers that are associated with HPV carcinogenesis rather than infection. These transformation specific biomarkers would be useful for studies of disease progression, prevention, and/or response to therapy. We used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines

Methods

Results

Conclusion