A molecular rotor thioflavin T (ThT) is usually used as a fluorescent ligand specific for G-quadruplexes. Here, we demonstrate that ThT can tightly bind non-G-quadruplex DNAs with several GA motifs and dimerize them in a parallel double-stranded mode, accompanied by over 100-fold enhancement in the fluorescence emission of ThT. The introduction of reverse Watson–Crick T-A base pairs into these dimeric parallel-stranded DNA systems remarkably favors the binding of ThT into the pocket between G•G and A•A base pairs, where ThT is encapsulated thereby restricting its two rotary aromatic rings in the excited state. A similar mechanism is also demonstrated in antiparallel DNA duplexes where several motifs of two consecutive G•G wobble base pairs are incorporated and serve as the active pockets for ThT binding. The insight into the interactions of ThT with non-G-quadruplex DNAs allows us to introduce a new concept for constructing DNA-based sensors and devices. As proof-of-concept experiments, we design a DNA triplex containing GA motifs in its Hoogsteen hydrogen-bonded two parallel strands as a pH-driven nanoswitch and two GA-containing parallel duplexes as novel metal sensing platforms where C–C and T–T mismatches are included. This work may find further applications in biological systems (e.g. disease gene detection) where parallel duplex or triplex stretches are involved.

Here, we for the first time demonstrated thioflavin T (ThT) as an efficient fluorescent ligand for 27-mer ATP-binding aptamer (ABA27), providing a novel signal readout mode for label-free selective ATP detection. ABA27 can promote the fluorescence emission of ThT with an unprecedentedly high efficiency, attributed to the specific structure of ABA27 rather than the G-tracts. Polyacrylamide gel electrophoresis, fluorescence spectroscopy, and fluorometric titration reveal that ThT interacts with ABA27 with a lower binding affinity (Kd ~89 μM) than ATP, which allows ATP to easily compete with ThT for the DNA binder. In the presence of ThT, adding ATP induces ABA27 to undergo a structural change, thereby not favoring the binding to ThT, verified by circular dichroism and UV-Vis absorption spectroscopy. As a result, the fluorescence intensity of ThT decreases dramatically, enabling the sensitive detection of ATP with high selectivity over other analogs. Such a sensing strategy may make ThT able to serve as a facile signal reporter for DNA nanomechanical devices fueled with ATP. Graphical Abstract The principle of the displacement of ThT by ATP.

We report a new signal readout mechanism for DNA molecular sensing devices using ligand-free fluorogenic G-quadruplexes in which the propeller-like loops are distinguished from the diagonal and lateral loops with incorporated 2-aminopurine (2-AP, a fluorescent analogue of adenine). We study the fluorescence behavior of looped-out 2-AP in duplexes and G-quadruplexes and demonstrate that it shows better fluorescence properties in shorter loops. In particular, 2-AP in the propeller-like loops of parallel or hybrid G-quadruplexes displays a perfect fluorescence emission whereas that in the diagonal and lateral loops does not. This loop-environment-sensitive feature allows 2-AP to probe the propeller-like loops of G-quadruplexes, illustrated by an ion-tuned allosteric G-quadruplex FG9A and a (3 + 1) hybrid human telomeric DNA. In the presence of K+, FG9A folds into a parallel structure where 2-AP is in the propeller-like loops and shows a high fluorescence signal, which can probe K+ concentrations down to 25 μM. Upon addition of Pb2+, the folded FG9A converts into an antiparallel structure which is revealed by a sharp decrease in 2-AP fluorescence, which can easily be reset with EDTA. This process is utilized to reversibly sense Pb2+ with a detection limit of 100 nM. Furthermore, its ability to probe the propeller-like loops may allow 2-AP to identify the folding topologies of unknown G-quadruplexes in human gene regions.