A Single lncRNA-Initiated Multiple Rolling Circle Amplification Based on Proximity Hybridization for One-Step Ratiometric Fluorescence Detection of lncRNAs in Breast Cancer

Long noncoding RNAs (lncRNAs) orchestrate critical control over gene expression dynamics, and their aberrant expression is a well-documented driver of malignant tumor initiation and progression. Herein, we developed a single lncRNA-initiated multiple rolling circle amplification (RCA) method with simple operation, rapid detection, high sensitivity, and excellent stability for one-step detection of lncRNAs in breast cancer. In this approach, we constructed a specialized primer that cannot hybridize to the circular template under conventional conditions. The target lncRNA (mammalian metastasis-related lung adenocarcinoma transcript 1, MALAT1) acts as a structural trigger to induce proximity hybridization between the primer and circular template, enabling native lncRNA to directly initiate RCA without reverse transcription or ligation pretreatments. Shortly after RCA initiation, the polymerase displaces the intact lncRNA from the template. The liberated target can then be repeatedly recycled to trigger new rounds of RCA, enabling a single lncRNA molecule to drive cascade amplification and drastically boost both signal efficiency and detection sensitivity. Ultimately, the Cy3-modified donor probe and Cy5-modified acceptor probe hybridize with the RCA product, thereby establishing an effective fluorescence resonance energy transfer (FRET). Benefiting from the unique mechanism where a single target can trigger multiple RCA events, this method possesses ultrafast reaction speed (20 min) and excellent sensitivity (limit of detection = 0.283 aM). This method not only allows for the discrimination of lncRNA expression levels among various cell lines but also realizes sensitive quantification of lncRNA in single MCF-7 cells. Most critically, it effectively discerns the expression differences of lncRNA between clinical breast cancer tissues and normal tissues within just 20 min, providing a robust technical tool for biological research, clinical point-of-care testing (POCT) diagnosis, and precision oncology.