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

Abstract

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 hu- mans. In this study, the artificial nucleic acid ap- tamers targeting α-amanitin were screened by Sys- tematic Evolution of Ligands by Exponential Enrich- ment (SELEX) in vitro, in order to develop an analyt- ical 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 detect- able concentration of aptamer H06 for α-amanitin was 8 ng/mL. The circular dichroism (CD) spectros- copy experiment indicated aptamer H06 forms a stem -loop and intramolecular G-quadruplex and it can stable exist in binding buffer and PBS buffer. Moreo- ver, 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