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RT-RPA-assisted scaffold RNA transcription amplification activation Cas12a trans-cleavage strategy for one-pot miRNA
Cheng-Jun Zha1, Hong-Shuai Zhang2, Sheng He3
1Key Laboratory of Environmentally Friendly Chemistry and Application of Ministry of Education, Key Laboratory for Green Organic Synthesis and Application of Hunan Province, College of Chemistry, Xiangtan University, Xiangtan, 411105, China.
Talanta
|April 3, 2025
Summary
We developed a novel ScRNA-TAAT strategy for rapid and highly sensitive microRNA detection. This method achieves attomolar detection limits in under an hour, suitable for point-of-care diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Diagnostics
Background:
- CRISPR systems, particularly split CRISPR RNA (crRNA), are vital for precise nucleic acid identification in molecular diagnostics.
- Current methods for microRNA (miRNA) detection often face limitations in sensitivity, speed, or complexity.
Purpose of the Study:
- To introduce a novel, one-pot strategy named Scaffold RNA Transcription Amplification Activation Cas12a Trans-cleavage (ScRNA-TAAT) for highly sensitive and rapid miRNA detection.
- To demonstrate the integration of ScRNA-TAAT into a visual lateral flow assay for point-of-care applications.
Main Methods:
- Utilized reverse transcription-recombinase polymerase amplification (RT-RPA) to amplify miRNA into double-stranded DNA with a T7 promoter.
- Developed a scaffold RNA transcription amplification system involving a split T7 promoter and a three-way linker.
- Assembled a Cas12a-based ribonucleoprotein (RNP) complex using scaffold RNA and DNA-RNA hybrids, mimicking split crRNA function for trans-cleavage activity.
- Integrated the ScRNA-TAAT assay with lateral flow analysis for visual detection.
Main Results:
- Achieved miRNA detection within 50 minutes with an ultra-low detection limit of 2.66 attomolar (aM).
- Demonstrated trans-cleavage activity of the assembled Cas12a RNP complex comparable to wild-type Cas12a RNP.
- Successfully applied the ScRNA-TAAT strategy to complex biological samples like serum and cell lysates, showing high specificity and universality.
- Developed a visual detection assay using lateral flow strips.
Conclusions:
- The ScRNA-TAAT strategy offers a simple, sensitive, and adaptable platform for miRNA detection.
- This method holds significant promise for advancing molecular diagnostics, particularly for point-of-care testing requiring high sensitivity.
