Aptamer-Braked Multi-hairpin Cascade Circuits for Logic-Controlled Label-Free In Situ Bioimaging

As a common hairpin-based amplification strategy, catalytic-hairpin assembly (CHA) has been widely used to construct various DNA circuits for biosensing and imaging. However, the hairpin substrates can potentially react without catalysts and result in circuit leakage, which may be quite severe in a CHA reaction consisting of three or four hairpins due to the formation of stable three-/four-way junction product. To circumvent this problem, here we introduce a well-designed ATP aptamer as a DNA brake into a four-hairpin cascade circuit, where the triggering toehold is blocked by the aptamer brake and thus the circuit leakage decreases dramatically. Such an aptamer-braked DNA circuit is then employed to build an AND logic gate in response to multiple external stimuli in acidic cell membrane microenvironments. Induced by a bimolecular i-motif that binds thioflavin T (ThT), the dimerization of a four-way junction in situ assembled on the cell surface is accomplished, enabling the logic-controlled cell membrane imaging in a label-free manner. Our design would be applicable to other hairpin-based amplification strategies and may find more applications in the construction of multiresponsive DNA cascade circuits in complex living systems.