Dynamic Monitoring of MicroRNA–DNA Hybridization Using DNAase-Triggered Signal Amplification

Dynamically monitoring microRNA (miRNA)–DNA reactions is critical for elucidating various biological processes. However, traditional strategies fail to capture this dynamic event because the original targets are preamplified. In the present study, we developed an amplification-free strategy for real-time monitoring of miRNA–DNA hybridization that integrates the advantages of both duplex-specific nuclease (DSN)-triggered signal amplification and single-stranded DNA probe coating facilitated by reduced graphene oxide. DSN-mediated miRNA recognition was found to consist of two phases: hybridization and hybridization cleavage. In the presence of miRNA and DSN, hybridization of a 22-mer miRNA–DNA could be completed within 7 min by observing the angle increase in a surface plasmon resonance (SPR) biosensor. The subsequent hybridization-cleavage process could be visualized as a gradual SPR angle decrease that occurred until all coated probes were hydrolyzed. In addition, for miRNA-21 detection, the proposed linear signal amplification assay demonstrated a sensitivity of 3 fM over a dynamic range of 5 orders of magnitude

High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·μL–1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics