One-pot dual-toehold RCA-Cas12a biosensor driven by a preassembled three-arm toehold-gated DNA template for sequence-selective miRNA liquid biopsy.

MicroRNAs (miRNAs) in blood are promising liquid biopsy biomarkers, yet their short length, low abundance, and high intra-family homology hinder sensitive and specific detection. Combining rolling circle amplification (RCA) with CRISPR-Cas12a enables isothermal detection, but existing methods typically depend on auxiliary enzymes or in-assay ligation and rarely encode sequence discrimination within the template itself. Here, we report a one-pot dual-toehold RCA (dtRCA)-Cas12a biosensor driven by a preassembled three-arm toehold-gated (3TG) DNA template for ultrasensitive and selective miRNA detection. The 3TG template adopts a three-arm dumbbell conformation, eliminating the need for a ligase during the assay, and presents two target-complementary toehold domains with a Cas12a-recognition sequence. Target binding triggers strand displacement, initiating dtRCA via a single polymerase. The resulting amplicons activate Cas12a trans-cleavage for fluorescence or lateral flow assay (LFA) readouts. Crucially, a single-base mismatch within the toehold suppressed amplification, whereas a topology-matched circular template lacking the toehold gate failed to distinguish the target, demonstrating that selectivity arises from the template structure. The one-pot dtRCA-Cas12a system achieved attomolar sensitivity, detecting miR-21, miR-375, and let-7a at 2.5, 114.9, and 8.0 aM, respectively. The paper-based LFA maintained femtomolar sensitivity and enabled an instrument-light readout. In plasma, this platform discriminated breast cancer patients (n = 17) from healthy donors (n = 10) with AUC values of 0.97-0.98. Three-marker classification demonstrated robust performance in leave-one-out cross-validation and correctly classified 30 samples in an independent validation cohort, showing performance comparable to RT-qPCR. By embedding selectivity into a preassembled template, this 3TG-driven dtRCA-Cas12a platform provides a highly sensitive and specific strategy for multi-marker miRNA analysis with simplified readout.