FEN1-Gated Rolling Circle Amplification Achieves Single-Base Mismatch Discrimination for Breast Cancer Biomarker Analysis

Rolling circle amplification (RCA) is a powerful isothermal nucleic acid amplification technique, yet its specificity is often compromised because padlock probes can be circularized even with imperfectly matched targets, leading to false-positive signals. Herein, we report a strand displacement-assisted FEN1 cleavage strategy to substantially improve the specificity of RCA. A dumbbell-shaped padlock probe is designed to generate dual 5' flaps only upon perfect hybridization with the target. Flap endonuclease 1 (FEN1) specifically recognizes the three-base overlap structure and cleaves the 5' flaps, enabling stringent single-base mismatch discrimination over a recognition length exceeding 23 base pairs. After FEN1 cleavage, the padlock is circularized by T4 DNA ligase, followed by RCA triggered by primer-conjugated magnetic beads. The RCA products form G-quadruplex structures that bind thioflavin T (ThT) for fluorescence readout. The method achieves excellent sensitivity for breast cancer-related biomarkers, with detection limits of 0.61 fM for hsa-miR-2682 and 1.13 fM for hsa_circ_0131242, along with high specificity (single-base resolution), good reproducibility (CV < 5%), and satisfactory recovery (95.1-101.5%) in serum. Clinical validation using 15 breast cancer patient samples and 15 healthy controls shows that the method reliably recapitulates differential expression of hsa_circ_0131242, with results highly consistent with RT-qPCR. By overcoming the inherent specificity limitation of conventional RCA, this FEN1-assisted, strand displacement-enhanced strategy provides a sensitive, reliable, and versatile platform for RNA biomarker analysis, holding great promise for early diagnosis of breast cancer and other diseases.