5′ Dual-Overhang Short PAM-less dsDNA as Switchable Activators of Cas12a trans -Cleavage for Amplification-Free miRNA Detection

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.