Selection and characterization of DNA aptamer specially targeting α-amanitin in wild mushrooms

Abstract

α-amanitin is a polypeptide isolated from the fruiting body of Amanita exitialis. It is the main toxin in wild mushrooms and toxic, often lethal, in animal and humans. In this study, the artificial nucleic acid aptamers targeting α-amanitin were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in vitro, in order to develop an analytical tool for α-amanitin detection. The specificity of aptamer H06 with α-amanitin was confirmed using Enzyme-Linked OligoNucleotide Assay (ELONA) and Dot blot, and no non-specific was observed. Based on the ELONA platform, the minimum detectable concentration of aptamer H06 for α-amanitin was 8 ng/mL. The circular dichroism (CD) spectroscopy experiment indicated aptamer H06 forms a stem -loop and intramolecular G-quadruplex and it can stable exist in binding buffer and PBS buffer. Moreover, the affinity test showed a strong binding force between α-amanitin and the aptamer H06, with the dissociation constant (KD) of 37.5±5.135 nM. And the accurary of the ELONA assay based on aptame H06 was demonstrated in real mushroom samples. In summary, our data could demonstrate a possibility of the development of apta-based diagnostic platform and detection method for α-amanitin.