Characterizing Aptamer-Ochratoxin A Interactions; Preliminary Work Towards the Construction of an Aptasensor

Abstract

Ochratoxin A is a secondary metabolite of Fungi that is correlated to kidney and liver damage, along with immune suppression, developmental defects, and cancer progression. Often, Ochratoxin A can be found as a contaminant of foods like barley, oats, rye, wheat, coffee beans, and wine. Given its many negative qualities, it is necessary to identify Ochratoxin A, to ultimately remove it from common household consumables. Currently, antibodies are used to identify Ochratoxin A, however, an alternative detection method employs aptamers in conjunction with a detection method like chemiluminescence or fluorescence (among others). Aptamers are single-stranded nucleotide sequences that bind to a specific ligand. In this study, a previously characterized aptamer that detects Ochratoxin A is used, and the aptamer-toxin interactions were assessed, with the intention of adding a DNAse sequence to the oligonucleotide, to enable toxin-detection. Native and denaturing gels were used to determine whether aptamer-toxin interactions alter the nucleotide’s running speed – no significant results were acquired. Similarly, the aptamer was titrated with Ochratoxin A (0X, 2.5X, 5X, 10X, and 20X) and run on a native gel; even the largest concentration of Ochratoxin A (20X) did not change the nucleotide’s running speed. Next, an absorption spectrum was produced using the toxin titration concentrations - results were inconclusive. DMS probing was performed using the toxin titration concentrations to characterize the binding of guanine residues within the aptamer to Ochratoxin A. With the aptamer-toxin interactions characterized, future research can construct the aptamer-DNAse oligonucleotide and test its ability to detect the toxin’s presence.

Department: Biology 

Faculty Mentor: Dr. Nina Bernstein