Dynamic DNA assembly-assisted CRISPR/Cas12a system for lung cancer-associated miRNA analysis

Serum-derived tumor-associated microRNAs (miRNAs) have emerged as clinically valuable biomarkers for early cancer detection and prognostic evaluation. The development of robust analytical platforms enabling ultrasensitive miRNA quantification remains an urgent priority in molecular diagnostics. Herein, a well-designed functional hairpin DNA template (H) combined with the CRISPR/Cas12a system was proposed as a novel strategy for ultrasensitive detection of lung cancer-associated miRNAs. The H-sequence undergoes a topological transition upon specific recognition of the target miRNA, initiating an isothermal exponential amplification reaction (iEXPAR) that continuously releases amplicons. These amplicons, in turn, activate the CRISPR/Cas12a system, resulting in signal amplification. This approach achieves a linear detection range from 20 fM to 2 nM, with an impressive detection limit as low as 26 fM. Due to the programmability of DNA sequences, this strategy holds great potential for the sensitive detection of a wide range of other nucleic acid targets. Herein, we developed a novel strategy for ultrasensitive detection of lung cancer-associated miRNA-21 using a functional hairpin DNA template (H) combined with the CRISPR/Cas12a system. The process begins with the specific recognition of target miRNA by the H-sequence, triggering a topological transition that initiates an isothermal exponential amplification reaction (iEXPAR). This reaction continuously generates amplicons, which activate the CRISPR/Cas12a system, leading to significant signal amplification. The method demonstrates a linear detection range from 20 fM to 2 nM, with a remarkable detection limit of 26 fM.