A programmable quantum dot nanosensor guided by three-way junction skeleton-mediated cascade signal amplification for sensitive detection of circRNAs in breast cancer

Circular RNAs (circRNAs) are endogenous covalent circular molecules that can regulate various biological processes in eukaryotes, and their dysregulation is associated with tumorigenesis. Herein, we demonstrate the construction of a programmable quantum dot (QD) nanosensor guided by three-way junction (TWJ) skeleton-mediated cascade signal amplification for sensitive detection of circRNA in clinical breast tissues. The TWJ probe contains a TWJ primer and a TWJ template. The presence of circRNA can specifically hybridize with TWJ probe to form a stable TWJ skeleton, initiating cascade signal amplification to produce abundant linker probes. The resultant linker probes can bind with Cy5-modified signal probes and biotinylated capture probes to yield the sandwich duplexes. Eventually, the immobilization of sandwich duplexes on the 605QD surface yields the 605QD-dsDNA-Cy5 nanoassembly, resulting in effective fluorescence resonance energy transfer (FRET). This nanosensor is free from exogenous reverse transcription primers and complicated probes design, and it exhibits high amplification efficiency, single-base mismatch specificity, and near-zero background signal. It can detect circRNA with attomolar sensitivity, measure endogenous circRNA at the single-cell level, and discriminate circRNA level in breast cancer tissues from adjacent normal tissues. Moreover, this nanosensor can be expanded to detect other circRNAs (e.g., circHIPK3) by easily changing the corresponding target recognition sequences of TWJ probes, with potential applications in biomedical research and molecular diagnosis.