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Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different
Qian Liu1, Ting-Ting Pan1, Li-Juan Wang1
1School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China.
Abstract:
Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.
Insights
We developed a fast and sensitive method to detect circular RNAs (circRNAs) using CRISPR-Cas12a technology. This new technique accurately identifies circRNAs in various samples, aiding in disease progression classification.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Circular RNAs (circRNAs) are noncoding RNAs with unique circular structures.
- circRNAs play crucial roles in regulating gene expression at transcriptional and post-transcriptional levels.
Purpose of the Study:
- To develop a sensitive, specific, and rapid method for detecting circular RNAs.
- To enable single-molecule profiling of circRNAs for potential diagnostic applications.
Main Methods:
- A one-pot assay utilizing engineered DNA hairpins for signal pre-amplification.
- CRISPR-Cas12a system for signal generation through trans-cleavage amplification.
- Detection of circRNAs from RNase R-treated RNA samples, including clinical specimens.
Main Results:
- Achieved a highly sensitive detection limit of 1.07 aM.
- Demonstrated specificity with single-mismatch discrimination capabilities.
- Completed detection within a rapid 25-minute reaction time.
- Successfully classified disease progression using clinical samples.
Conclusions:
- The developed platform offers a sensitive, specific, and fast method for circRNA detection.
- This technique enables single-molecule profiling and has potential for clinical diagnostics.
- The method can be extended for the detection of other nucleic acid types.
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