Abstract

Precise and programmable regulation of CRISPR–Cas12a activity is essential for advancing controllable nucleic acid diagnostics, yet the structural determinants governing Cas12a activation by short PAM-less double-stranded DNA (dsDNA) remain largely unexplored. This study systematically investigates the effects of the terminal architectures of short PAM-less dsDNA on Cas12a trans -cleavage activity. By profiling a series of dsDNA constructs bearing distinct 5 ′ /3 ′ overhang configurations, a 5 ′ dual-overhang motif was identified as a highly effective structural inhibitor that suppresses Cas12a activation. Kinetic fluorescence assays combined with computational structural modeling indicated that this inhibition arises from steric constraints imposed by the 5 ′ terminal architecture. Leveraging this structure-guided regulatory mechanism, an amplification-free CRISPR–Cas12a assay was developed for the direct detection of oncogenic microRNAs miR-155 and miR-21, achieving femtomolar sensitivity without reverse transcription. The assay was further evaluated in human serum samples spiked with target miRNAs, supporting its proof-of-concept performance in a more complex matrix. Collectively, these findings highlight the potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity and provide useful insight for the design of CRISPR-based biosensing strategies.