Development of a colorimetric aptasensor for aflatoxin B1, featuring modular fluorescent oligonucleotide labeling

Aptamers are nucleic acid-based ligand binding molecules that are capable of strong and specific binding to small molecule, protein, or whole cell ligands. These versatile nucleic acids can be paired with visualization techniques to create sensors aptly named aptasensors. Our research project is aimed at developing a DNA-based aptasensor for the detection of aflatoxin B1 (AFB1) by pairing it with DNAzyme. For the purposes of our project, DNAzyme can be thought of as a DNA sequence that will fold into a G-quadruplex to catalyze an oxidation reaction ultimately producing a green colour from colourless reagents. To properly design our aptasensor, we are beginning with characterization steps. First, we ran a native gel to test for conformational change, but the results were inconclusive. Next, we plan to carry out DMS probing, which is a type of fingerprinting experiment that should elucidate the binding sites of AFB1 on our aptamer. For this, we require an end-labeling technique. Since our university is not equipped for radioactive end labeling, we decided to develop a modular fluorescent end labeling technique. This technique involves a 5’-fluorescently labeled “probe” molecule (14 mer) that is ligated to the aptamer by use of an adaptor oligonucleotide (complementary to both the probe and 3’ end of the aptamer), T4 PNK, and T4 DNA ligase. Based on preliminary results, the labeling was successful, we will examine whether it interferes with aptamer function during DMS probing. Once our aptamer is characterized, we intend to design a same strand split DNAzyme sensor. We will not know the exact mechanism until we have tested it, though we hope that we will see differential DNAzyme formation upon AFB1 binding, leading to the ability to differentiate between varying concentrations of AFB1 based on green colour change and making it a viable aptasensor.