Characterization of an Aptamer for AFB1:

An Attempt at Aptasensor Design and Modular End Labeling

Abstract

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 which are aptly named aptasensors. This research project was aimed at developing a colorimetric DNA-based aptasensor for the detection of aflatoxin B1 (AFB1) by pairing it with DNAzyme in a same-strand split design. To properly design the aptasensor, characterization experiments were carried out. These included a native gel to test for conformational change, UV absorption spectra, and DMS probing. The native gel and UV absorption spectra were low in resolution and did not provide valuable information regarding conformational change. DMS probing is a type of fingerprinting experiment that allowed for the elucidation of AFB1 binding sites on the aptamer. An end-labeling technique was required for the DMS probing. Since MacEwan University is not equipped for radioactive end labeling, a modular fluorescent labeling technique was developed. This technique involved a 5’-fluorescently labeled “probe” molecule 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. Overall, our end labeling technique was functional and DMS probing allowed for aptamer characterization, but time did not allow for the aptasensor to be designed and tested. Now that the aptamer has been characterized, aptasensor design and testing may be carried out in a future project and the end labeling technique may be used in future aptamer research.

Department: Honours Biology

Faculty Mentor: Dr. Nina Bernstein