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5' Dual-Overhang Short PAM-less dsDNA as Switchable Activators of Cas12a trans-Cleavage for Amplification-Free miRNA
Xiaohui Wang1,2, Shuxin Zhao3, Jiaye Jiang1,2
1School of Microelectronics, Shanghai University, Shanghai201899, China.
ACS Sensors
|July 13, 2026
Summary
Engineered DNA structures can control CRISPR-Cas12a enzyme activity for nucleic acid diagnostics. A 5
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR-Cas12a activity requires precise and programmable regulation for nucleic acid diagnostics.
- Structural factors influencing Cas12a activation by short, PAM-less double-stranded DNA (dsDNA) are not well understood.
Purpose of the Study:
- To systematically investigate how the terminal structures of short, PAM-less dsDNA affect Cas12a trans-cleavage activity.
- To develop a novel CRISPR-Cas12a-based biosensing strategy for microRNA detection.
Main Methods:
- Profiling of dsDNA constructs with varying 5'/3' overhang configurations.
- Kinetic fluorescence assays and computational structural modeling.
- Development of an amplification-free CRISPR-Cas12a assay for microRNA detection.
Main Results:
- A 5' dual-overhang motif was identified as a potent inhibitor of Cas12a activation.
- Inhibition is attributed to steric hindrance from the 5' terminal DNA architecture.
- The developed assay achieved femtomolar sensitivity for detecting oncogenic microRNAs miR-155 and miR-21 without requiring reverse transcription.
Conclusions:
- Terminally engineered PAM-less dsDNA can serve as a structural element to program Cas12a activity.
- This study offers insights for designing advanced CRISPR-based biosensing strategies.
- The developed assay demonstrates proof-of-concept performance in complex biological matrices like human serum.
