DNA Aptamer–Target Binding Motif Revealed Using a Fluorescent Guanine Probe: Implications for Food Toxin Detection

Abstract

DNA aptamers are single-stranded oligonucleotides that are generated by an in vitro selection method to bind targets with high affinity and specificity. Understanding molecular recognition by DNA aptamers is of fundamental importance in the development of biosensor applications. The small molecule ochratoxin A (OTA) is a fungal-derived food toxin, and OTA DNA aptamers have been established for the development of rapid detection platforms required for food safety. One such OTA aptamer (OTAA) is a guanine-rich DNA oligonucleotide that folds into an antiparallel G-quadruplex (GQ) upon OTA binding, although structural details of the GQ fold and its interaction with OTA are currently unknown. In the present study, the fluorescent nucleobase analogue, 8-thienyl-2′-deoxyguanosine (ThdG), was inserted into various G sites of OTAA to determine the probe impact on GQ folding and OTA binding affinity. Our results suggest that OTAA contains three lateral (l) loops connecting two stacked G-tetrads with an anticlockwise loop progression to afford a −(lll) GQ topology. The phenolic ring system of OTA undergoes π-stacking interactions with the G-tetrads of OTAA. Our results also demonstrate aptamer sites that can be modified with ThdG to afford a fluorescent light-up signal upon OTA binding.

Spectral-Phase Interferometry Detection of Ochratoxin A via Aptamer-Functionalized Graphene Coated Glass

“…While graphene provides strong adsorption of DNA by π-π stacking of nucleotides [ 37 ], the gold or glass substrates have weak binding energy to DNA, leaving it free-standing. Importantly, OTA aptamers are guanin-rich and have been shown to fold in the anti-parallel G-quadruplex after binding to the OTA [ 38 ]. Thus, upon OTA binding to the aptamer, the folded G-quadruplex will increase the effective thickness on the graphene layer.…”

Recent Progress and Development of G-Quadruplex-Based Luminescent Assays for Ochratoxin A Detection

“…A novel fluorescent nucleobase analog, 8-thiophene-2′-deoxyguanosine (ThdG), was inserted into the OTA aptamer to determine the effect of the probe on G4 folding and OTA binding affinity. Interestingly, the ThdG-modified aptamer sites can provide a fluorescent response after OTA binding (Fadock and Manderville, 2017 ). TiO 2 quenches non-covalent adsorption of FAM-labeled aptamers TiO 2 (Sharma et al, 2015 ).…”

An Electrochemical Sensor Based on Structure Switching of Dithiol-modified Aptamer for Simple Detection of Ochratoxin A

“…The addition of the target to the sensor induced the formation of G-quadruplex conformation, resulting in structural switching of the aptamer. 31 Furthermore, the MB molecule became closer to the electrode surface, generating a higher current than the background (step D). After the analysis, the sensing layer of the sensor was regenerated as described below.…”