A wide range of pathologies have been targeted with bimodular aptamers that contain both G-quadruplex (G4) and duplex motifs, while the structures and functions are poorly understood. G4-selective fluorescent dyes have served as facile tools to probe G4s, but not for bimodular aptamers, yet. Here, taking the 29-mer thrombin binding aptamer (TBA29) as an example, we demonstrated that 3,6-dimethyl-2-(4-dimethylaminophenyl)-benzothiazolium (ThT) was the most effective dye compared to NMM and PPIX in recognizing TBA29. Binding studies indicate that ThT recognized TBA29 via distinct buffer-dependent mechanisms. Specifically, ThT induced the formation of a bimolecular parallel G4 in cation-deficient buffer, showing 341-fold fluorescent enhancement. The competitive binding of thrombin disrupted the complex, leading to the monotonic fluorescence decrease. A similar mechanism was previously reported for the interaction between ThT and the 15-mer thrombin binding aptamer (TBA15). However, TBA29 bound with ThT in a more favorable state than TBA15, showing hyperchromic effects and two times stronger fluorescence enhancement. Differently, ThT bound with antiparallel TBA29/TBA15 in an intercalating/groove binding mode in 100 mM KCl, generating 181/28-fold fluorescence enhancement, respectively. These results revealed that ThT recognized both parallel and antiparallel G4s of TBA29 more efficiently than it recognized TBA15. The duplex structure of TBA29 may play an important role in its interaction with ThT. Our study broadens the application of ThT in screening G4 to bimodular aptamers and provides some insights into the structures of TBA29, along with the interaction between ThT and TBA29. Our study also is useful for the development of structure-switching-based biosensors using bimodular aptamers. Graphical abstract The buffer-dependent binding mechanisms of ThT with TBA29, and the competitive (top)/noncompetitive (bottom) binding of thrombin with TBA29-ThT complex.
Keywords: 29-mer thrombin binding aptamer; Bimodular aptamers; G-quadruplexes; Structure-switching aptamers; Thioflavin T; Thrombin.