Related Experiment Video
Updated: Sep 28, 2026
A Flow Cytometry-Based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer
Published on: September 13, 2022
Identification of a novel nucleolin-binding aptamer for specific tissue recognition and targeted drug delivery in
Xue Bai1, Xiao Li1, Zhixiang Huang2
1School of Basic Medical Sciences, State Key Laboratory of Metabolic Dysregulation & the Prevention and Treatment of Esophageal Cancer, Zhengzhou University, Zhengzhou, Henan, 450001, China.
Abstract:
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies, characterized by a poor prognosis largely attributable to the lack of early diagnostic markers and targeted therapeutic agents. Aptamers, screened through the Cell-based Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX), offer potential in the discovery of novel tumor biomarkers and the development of targeted cancer therapies. In this study, a novel aptamer, B7, was obtained through Cell-SELEX, demonstrating specific and high-affinity binding to ESCC KYSE30 target cells, whereas exhibiting no affinity for normal esophageal epithelial Het-1A cells. The B7 aptamer preferentially bound multiple ESCC cell lines and, when labeled with Cy5, enabled the recognition of clinical ESCC tissues via fluorescence imaging. Subsequent sequence optimization led to the development of a truncated variant, B7-6, which exhibited improved binding affinity. Pull-down assays combined with mass spectrometry identified nucleolin (NCL) as a candidate molecular target of B7-6, which was further validated by electrophoretic mobility shift assays, RNA interference, and competition binding assays. In vivo fluorescence imaging confirmed that B7-6 could target tumors in vivo. To demonstrate its therapeutic potential, a targeted delivery system, B7-6-FdU, was constructed by substituting all thymidines in B7-6 with 5-fluoro-2'-deoxyuridine (5FdU). This conjugate maintained strong target-binding ability and exhibited selective cytotoxicity against ESCC cells in vitro, whereas exhibiting reduced toxicity toward normal cells. In a xenograft mouse model, B7-6-FdU significantly suppressed tumor growth, producing the lowest mean tumor volume while maintaining stable body weight. Collectively, this study reports a novel NCL-binding aptamer, B7-6, which serves as a promising molecular tool for tissue recognition and targeted drug delivery in ESCC.
