Combined Target-Immobilized and Library-Immobilized SELEX for Selecting High-Affinity α-Amanitin Aptamers

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• Accidental ingestion of poisonous mushrooms, particularly Amanita species, leads to fatalities annually due to the absence of a specific antidote for alpha-amanitin.
• Early detection of alpha-amanitin in whole blood is crucial for timely intervention and preventing severe liver toxicity.
• Aptamers, as antibody mimics, offer potential for detecting small molecules like alpha-amanitin, but their selection is challenging due to the target's small size and limited binding sites.

Purpose of the Study:

• To develop an effective aptamer selection strategy for the small-molecule toxin alpha-amanitin.
• To achieve highly specific detection of alpha-amanitin in complex biological matrices like whole blood, overcoming interference from components such as human serum albumin (HSA).

Main Methods:

• Implemented a dual SELEX (Systematic Evolution of Ligands by Exponential Enrichment) approach, combining target-immobilized and library-immobilized strategies for aptamer selection.
• Utilized counter-selection techniques to eliminate aptamer sequences that bind to human serum albumin (HSA), thereby reducing non-specific binding and enhancing target specificity.
• Characterized the selected aptamers for their binding affinity and specificity towards alpha-amanitin.

Main Results:

• Successfully selected a high-affinity aptamer capable of specifically recognizing alpha-amanitin.
• The developed aptamer demonstrated nanomolar affinity for alpha-amanitin.
• The selection strategy effectively addressed challenges associated with small-molecule aptamer development and minimized interference from HSA.

Conclusions:

• The proposed aptamer selection method is effective for identifying high-affinity aptamers against small-molecule toxins like alpha-amanitin.
• The developed alpha-amanitin aptamer holds promise for sensitive and specific detection in whole blood, facilitating early diagnosis of poisoning.
• This approach advances the development of diagnostic tools for toxin detection and management of poisoning cases.