Selection and targeting of EpCAM protein by ssDNA aptamer

Abstract

Aptamers are molecules that reveal highly complex and refined molecular recognition properties. These molecules are capable of binding with high affinity and selectivity to targets, ranging from small molecules to whole living cells. Several aptamers have been selected for targeting cellular proteins and they have also used in developing therapeutics and diagnostic strategies. Epithelial cell adhesion molecule (EpCAM) is considered as a cancer stem cell (CSC) biomarker and one of the most promising targets for aptamer selection against CSCs. In this study, we have developed a ssDNA aptamer with high affinity and selectivity of targeting the EpCAM protein extracellular domain. The SELEX technique was applied and the resulted sequences were tested on EpCAM-positive human gastric cancer cell line, KATO III, and the EpCAM-negative mouse embryonic fibroblast, NIH/3T3 cells. Ep1 aptamer was successfully isolated and showed selective binding on EpCAM-positive KATO III cells when compared to EpCAM-negative NIH/3T3 cells, as observed by the flow cytometry and the confocal imaging results. Additionally, the binding of Ep1 to EpCAM protein was assessed using mobility shifting assay and aptamers-protein docking. Furthermore, the binding affinity of Ep1 was measured against EpCAM protein using EpCAM-immobilized on magnetic beads and showed apparent affinity of 118 nM. The results of this study could suggest that Ep1 aptamer can bind specifically to the cellular EpCAM protein, making it an attractive ligand for targeted drug delivery and as an imaging agent for the identification of cancer cells.

In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers

“…Considering the above arguments (including experimental observations that EpCAM dimerizes in vivo using the TY loop), we argue that the monomer aptamer binding conformations proposed in Fig. 2 and found in the literature (38,39), which bind EpCAM at the TY loop dimer interface, should be discounted as not physiologically relevant. Although the experiments performed in both papers indicate the aptamers do bind to EpCAM (38,39), the proposed binding conformations are inconsistent with solved crystal structures of EpCAM (33,40).…”

“…2 and found in the literature (38,39), which bind EpCAM at the TY loop dimer interface, should be discounted as not physiologically relevant. Although the experiments performed in both papers indicate the aptamers do bind to EpCAM (38,39), the proposed binding conformations are inconsistent with solved crystal structures of EpCAM (33,40). These conclusions lead us to include the background sections of physiology and biochemistry and of therapeutic action in our aptamer design protocol presented in SI Appendix, Fig.…”

“…We further identified how the designed RNA aptamer binds to the in vivo-relevant and in vitro-relevant form of EpCAM: the EpCAM dimer. Similar to other studies (38,39), we used docking to predict RNA binding conformations onto the EpCAM monomer. However, the predicted docking conformations showed strong binding preference for the TY loop of the EpCAM monomer.…”

Detection of Gold Nanoparticles Aggregation Using Light Scattering for Molecular Sensing

“…The amount of the aptamer adsorbed on the AuNPs was quantified as previously described. 23 Briefly, the prepared apt-AuNP solution (200 μL) was centrifuged (25 C, 8000 rpm, 15 min). The supernatant was removed, and 10% 2-ME (20 μL) was added to remove the Th-apt adsorbed on the AuNPs.…”

Utilizing Peptide Ligand GPCRs to Image and Treat Pancreatic Cancer

“…Aptamers can be attached to a variety of payloads, including small interfering RNAs (siRNAs), cytotoxic drugs, or nanoparticles, which improves the selective delivery and efficacy of the cargo [ 79 , 80 , 81 , 82 ]. Tumor-targeting aptamers have been selected for cell-adhesion molecules such as EpCAM, tyrosine kinase receptors, mucins, and other cell-surface proteins [ 83 ]. For example, EGFR-targeted aptamers conjugated to gold nanospheres have been successfully used to image head and neck tumors [ 84 ].…”