Synchronous dual-Cas12a activation via dimeric palindromic hairpin-programmed cascade amplification for ultrasensitive molecular diagnostics.

Integrating isothermal nucleic acid amplification with CRISPR/Cas12a trans-cleavage has emerged as a powerful strategy for ultrasensitive molecular diagnostics. However, most systems depend on multiple probes, separated amplification modules, or complex probe networks, increasing design complexity, optimization burden, and instability. Herein, we report a dimeric palindromic hairpin-programmed cascade amplification strategy for synchronous dual-Cas12a activation and ultrasensitive molecular diagnostics. The distinctive feature of this design lies in the construction of a single self-dimerizing palindromic hairpin (PaH) probe integrating target recognition, primer-initiated extension, nicking-site formation, cyclic trigger generation, and palindrome-directed trigger assembly. miRNA-155 was selected as a model biomarker to initiate the single-probe amplification process. Upon target recognition, the dimeric palindromic hairpin probe undergoes Phi29 polymerase-mediated extension and Nt.BbvCI-assisted cyclic nicking, continuously generating palindromic trigger strands. These triggers undergo intermolecular hybridization and polymerase-driven elongation to produce extended duplex structures containing dual crRNA-binding sites, synchronously activating two Cas12a complexes from one cascade amplification output. Owing to this architecture-embedded cascade amplification and dual-Cas12a trans-cleavage mechanism, provided a quantitative range of 1 fM to 1 nM, with a calculated detection limit of 55 aM. The assay exhibited high sequence specificity and, in a preliminary proof-of-concept evaluation using total miRNA extracts from a small cohort of healthy individuals and breast cancer patients, generated significantly different fluorescence responses between the two groups. By integrating multiple amplification and signal-transduction functions into a single probe architecture, this work provides a compact framework for constructing high-gain CRISPR/Cas12a-based biosensing systems.