Development of folding-based and signal-amplified fluorescence aptasensors using G-quadruplex quinclorac aptamer variants engineered via exonuclease digestion and circular dichroism spectroscopy

The lack of inherent structure-switching functionality and inadequate affinity in aptamers hinders the construction and application of aptasensors. While exonuclease digestion-based strategies have been employed to engineer small-molecule aptamers with the structure-switching functionality and construct dual-exonuclease (exonuclease I and exonuclease III) digestion-based aptasensors, such studies targeting G-quadruplex (G4) aptamers remain scarce. This study focused on the exonuclease digestion and circular dichroism spectroscopy analysis of a G4 quinclorac (QNC) aptamer, Qapt-51, intending to design a functionalized QNC aptamer and a universal signal amplification strategy for constructing folding-based and sensitive dual-exonuclease digestion-based aptasensors, respectively. The exonuclease digestion results demonstrated that QNC-binding Qapt-51 inhibited the dual-exonuclease digestion. Combining the results from the circular dichroism spectroscopy, we further proposed a digestion mechanism for Qapt-51 involving a G4 conformational switch. Inspired by this mechanism, a functionalized Qapt-51 variant (Qapt-45A) was rationally designed to construct a simple and rapid folding-based fluorescence aptasensor. Additionally, we developed an exonuclease III- and phosphorodiamidate morpholino oligomer-assisted fluorescence signal amplification strategy, which can work in the dual-exonuclease digestion system. Leveraging this strategy integrated with the dual-exonuclease digestion of another Qapt-51 variant (AQ), a homogeneous signal-amplified fluorescence aptasensor was constructed to sensitively detect QNC without requiring the functionalized aptamers. These two aptasensors achieved detection limits of 560 ng/mL and 8.5 ng/mL for QNC. They successfully detected QNC in rice and river water samples with recoveries of 108.6%-87.5%. We hope that the successful development of these two aptasensors can provide valuable insights and references for the broader construction of G4 aptamer-based sensing systems.