Simple yet Sensitive MicroRNA Detection Using Allosteric Probe-Initiated Triple Amplification and Cas13a/crRNA-Based Amplification Reaction

Abstract

MicroRNAs (miRNAs) have emerged as promising biomarkers for early diagnosis and management of degenerative disc disease (DDD); however, their low abundance and high sequence homology pose significant challenges for clinical detection. Herein, we develop a novel, highly sensitive miRNA detection platform by integrating an allosteric probe-initiated triple-cycle amplification strategy with the CRISPR-Cas13a/crRNA system. The designed allosteric probe undergoes a conformational switch upon target miRNA binding, triggering successive enzymatic amplification steps, including polymerase-mediated extension, nicking enzyme-driven recycling, and T7 RNA transcription, to generate numerous single-stranded RNA activators. These activators specifically recruit the Cas13a/crRNA complex, unleashing its collateral cleavage activity to degrade reporter RNAs and produce amplified fluorescence signals. This method demonstrates a wide dynamic range from 1 fM to 100 pM and achieves an ultra-low detection limit of 548 aM. Notably, the approach exhibits excellent specificity, distinguishing target miRNA-155 from closely related variants and non-target miRNAs. Its operational simplicity, rapid turnaround (60 min), and robustness in serum samples highlight strong potential for clinical translation. By combining catalytic allosteric probing with CRISPR-based signal amplification, this work provides a versatile and powerful tool for miRNA quantification, paving the way for early, minimally invasive diagnosis of degenerative disc diseases and other miRNA-associated pathologies.