Characterization and Application of Novel Structure Switching Aptamers for Aflatoxins

Abstract

Aflatoxins are a class of mycotoxins that are of great agro-economic importance.Their severe acute and chronic health effects have made them of great concern from a public health perspective as well. Due to their health implications, and large economic burden, aflatoxins have become the most studied group of mycotoxins. A specific interest in rapid, robust, and cost effective, on-site sensing platforms for mycotoxin contaminants has been shown. Aptamers are functional oligonucleotide sequences that are selected to bind a cognate ligand with high affinity and selectivity. Novel structure switching aptamer candidates for total aflatoxin were previously selected. The selection was performed using two template designs. Electrophoretic mobility shift assays, melting temperature studies, and circular dichroism along with predictive structural characterization techniques were used to help elucidate the aptamer candidate's structural biases for G-quadruplex formation. Two candidates were determined to bind aflatoxin B1 (AFB1) with high affinity using microscale thermophoresis. Sequences DZA3 and 3MR5 had reported dissociation constants (Kd) for AFB1 of 42.1 ± 23.8 nM and 1.4 ± 6.7 μM, respectively. Using the AFB1 binding aptamers, progress has been made towards developing FRET-based signalling sensors that leverage the structure-switching properties of the aptamer.iii

Fluorescence Aptasensor Development for Aflatoxin B1

“…This approach was recently used to find aptamers that had strong affinity for aflatoxins. 41 The aptamer template contained a fixed central region and randomized flanking regions, followed by PCR primer binding region. The DNA library was immobilized onto a solid support through a 12-base complementary region incorporated into the fixed central domain.…”