Here, fluorescent molecular rotors are employed to develop a new type of high-performance FRET system with large Stokes shift, high photostability, and pH insensitivity, showing great promise for use in proximity-dependent DNA aptasensors. Two carboxylated benzothiazole-based molecular rotors are synthesized, displaying bright green and red fluorescence once labeled to DNA. In the proximity state, an efficient FRET occurs between the two dyes, comparable to that of the most commonly used Cy3/Cy5 pair. Similar phenomena are also observed if naphthothiazole-based analogues are adopted. Our developed FRET pair is then attached to the two parts of a split ATP aptamer in a dimeric DNA nanoscaffold controlled by a bimolecular i-motif. In this way, a pH-switched proximity-induced fluorescent ATP aptasensor is constructed, with good sensitivity, selectivity, and reconfiguration. Furthermore, by altering the linker length of the switching unit, the proximity effect is investigated systematically, providing new insight into DNA proximity reactions and their roles in some physiological processes.
Keywords: ATP aptasensor; DNA assembly; FRET; molecular rotor; proximity effect.