Dataset Information

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.

SUBMITTER: Alshaer W

PROVIDER: S-EPMC5731996 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI). However, the current program for NGAL detection is not extensively applied in clinics due to the high expense of antibodies. Nucleic acid aptamers are single-strand DNAs or RNAs which could bind to targets with high specificity and affinity, and they have been widely used in the diagnosis and therapy for multiple diseases. It is valuable for us to develop a new method for NGAL detection using aptamers instead of antibodies to achieve increased efficiency and decreased cost.MethodsNucleic acid aptamers against NGAL were obtained after SELEX process using magnetic beads, and an enzyme-linked aptamer analysis (ELAA), which can be widely used in clinical diagnosis at low cost, were successfully established. The feasibility of ELAA was further validated with urine samples harvested from 43 AKI patients and 30 healthy people.ResultsThree candidate aptamers, including NA36, NA42 and NA53, were obtained after 8 rounds of SELEX process with magnetic beads and verified by quantitative polymerase chain reaction (qPCR), and the Kd value of each aptamer was 43.59, 66.55 and 32.52 nM, respectively. Moreover, the linear relationship was consistent at the range of 125-4000 ng/mL, and the detection limit of ELAA assay was 30.45 ng/mL. We also found that NGAL could be exclusively detected with NA53, and no cross-reaction between NA53 and human albumin or globulin occurred, the coefficient of variation (CV) between inner-plate and inter-plate was less than 15%, and the recovery rate was between 80 and 110%. Moreover, the sensitivity and specificity of ELAA assay in this study are 100% and 90%, respectively. Consistently, these results could also diagnose whether the occurrence of AKI in lots of patients, which has been demonstrated with the ELAA method we established after using NA53.ConclusionsTaken together, NA53, the best candidate aptamer targeting NGAL protein, can be applied in clinical testing.

Dataset Information

Selection and targeting of EpCAM protein by ssDNA aptamer.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets