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Specific and sensitive fluorescence anisotropy sensing of guanine-quadruplex structures via a photoinduced electron transfer mechanism.

Fluorescence anisotropy (FA) is a homogeneous, ratiometric, and real-time analytical technology. By selective labeling of a guanine (G)-quadruplex motif with tetramethylrhodamine (TMR), here, it is established that a large reduction in FA response can be specifically associated with the unfolding → folding transition of G-quadruplex structures. On the basis of fluorescence intensity, polarization and lifetime analysis, and molecular docking simulation, the mechanism was found to be that the labeled fluorophore (TMR) can intramolecularly interact with adjacent G bases in an unfolded G-quadruplex motif, which allows for the photoinduced electron transfer (PET) occurring between the fluorophore and G bases, leading to a short fluorescence lifetime. Upon the folding of the motif to form a stable G-quadruplex structure, the intramolecular interactions and the concomitant PET could be eliminated with an increased fluorescence lifetime, leading to a large reduction in the FA response. On the basis of this mechanism, a novel, specific, and sensitive FA approach was developed for the detection of biologically and functionally important G-quadruplex structures. The approach is examined and validated using one normal G-quadruplex motif, five mutants, and six small cations and is potentially applicable to the study of G-quadruplexes at single molecule level, ligand screening, profiling of highly ordered DNA nanostructures, and biosensing.