A triangular split-DNAzyme scaffold for high-contrast microRNA imaging in living cells

The 10-23 DNAzyme is a potent catalytic nucleic acid for biosensing; however, traditional single-blocker designs suffer from high background signal leakage due to spontaneous thermal “breathing.” To overcome this, we engineered a triangular DNA probe-DNAzyme (TDPzyme) that spatially separates the enzyme components onto a rigid 3D scaffold. By splitting the binding arms and catalytic center, the TDPzyme functions as a fault-tolerant logic “AND” gate, strictly requiring the simultaneous displacement of multiple blocker strands by the target miR-21. This architectural constraint physically shields the probe from nuclease degradation and virtually eliminates false-positive activation, yielding a near-zero background state. Functionalized with a Cy3-BHQ2 pair and an AS1411 aptamer for targeted, carrier-free cellular delivery, the TDPzyme achieved an approximately 18-fold enhancement in sensitivity over conventional closed-arm designs, establishing a linear range of 0.1 nM to 40.0 nM and a limit of detection of 50.3 pM (3σ). Furthermore, time-lapse dynamic imaging demonstrated that the TDPzyme enables highly reliable, high-contrast discrimination of miR-21 expression between tumor and normal cells. Ultimately, the TDPzyme design offers a robust, structurally controllable platform for developing highly sensitive, low-background biosensors applicable to demanding live-cell imaging and precision molecular diagnostics.