•This novel strategy is based on ligand-responsive G-quadruplex formation.•The assay is label free for rapid DNA detection.•This probe is simple and cost-efficiency in design and operation.A facile and label-free assay with label-free molecular beacons (MBs) and fluorescent dye N-methyl mesoporphyrin IX (NMM) was developed for the detection of specific single-stranded DNA sequences. It was demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 20 bases) of RNA fragment of human immunodeficiency virus (HIV) as model systems. In the absence of target DNA, the MBs were in the stem-closed form, the G-quadruplex structure could not form and the fluorescence signal of NMM was very low. In the presence of target DNA the MBs turned “Off” to “On”, thus promoting the formation of G-quadruplex which could greatly enhance the fluorescence of NMM. This biosensor was simple in design, fast in operation, and more convenient and promising than other methods. It took less than 30 min to finish and its detection limit was 1.4 nM. No sophisticated experimental techniques or chemical modification for DNA sequences were required. This new approach could be widely applied to sensitive and selective nucleic acids detection.

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a “plug-and-play” fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil–DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil–DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.