Study Of DNA Aptamers Targeting α-amanitin

Amanita is the main toxin produced by wild mushrooms, which is usually caused by the poisoning and death of human and animal. Epecially in China, there are a lot of consumers have died because of poisonous mushrooms every year. There are about 2000 kinds of edible fungi can be identified in the world. There are 50 kinds of them are poisonous. a-Amanitin is the main toxin in mushrooms of the Amanita genus and it is often involved in animal and human intoxication or death. While,5 mg of a-amanitin can cause death. It is an octapeptide compound. Besides, it is a specific inhibitor of eukaryotic RNA polymerase II in biological research.At present, some direct protein detection assays are available for detection of a-amanitin. Most of them have certain limitation because of the complex operation, expensive instruments or easy cross-contamination. In recent years, aptamers have been investigated as an alternative for bio-recognition ligands. Aptamers are artificial nucleic acid ligands that can bind to target molecules with high affinity and specificity. These targets rang from large molecules such as protein to simple organic small molecules like ATP, dyes, amino acids or small cations. The aptamers are generated by an in vitro selection process called systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves a series of enrichment cycles and counter selection based on repetitive binding that ultimately selects for a group of aptamers binding to the targets. Aptamers can specifically recognize the targets or regulate their functions. They have numerous advantages over antibodies because of their higher specificity, convenient synthesis, no immunogenicity reaction, and long-term stability.In this study, two aptamers targeting α-amanitin were cloned affinitively and specifically in order to develop an analytical tool for a-amanitin detection. An ssDNA random library was subjected to 12 rounds of selection against a-amanitin according to the protocol of systematic evolution of ligands by exponential enrichment (SELEX) method, Affinity filler epoxy-activated Sepharose 6B was used as screening medium. The twelfth round of screening product was cloned and sequenced. The secondary structure of the obtained 12 aptamers revealed stem loop and G-quadruplex were the main structure forms. It forecasted that they were the basic structure of specific binding between aptamer with a-amanitin by circular dichroism. A new technology called ELONA was built in this study based on selected aptamers target for a-amanitin. The results of aptamers specificity and sensitivity showed that E06 and H06 were the best ones by ELONA. Our research laid the foundation for detecting a-amanitin in mushroom quickly and precisely.