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Structural and Kinetic Profiling of Rolling Circle Amplification via Solid-State Nanopore Sensing Using miR-21 as a
Kawin Loha1, Thitikorn Boonkoom2, Harit Pitakjakpipop2
1School of Biomolecular Science and Engineering (BSE), Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand.
ACS Sensors
|September 16, 2025
Summary
This study combines Rolling Circle Amplification (RCA) with nanopore sensing to observe DNA amplification at the single-molecule level. The novel label-free method reveals real-time changes in DNA structure during amplification, aiding diagnostic tool development.
Area of Science:
- Molecular Biology
- Nanotechnology
- Biophysics
Background:
- Rolling Circle Amplification (RCA) is a key isothermal nucleic acid amplification method.
- Molecular diagnostics require sensitive and specific detection methods.
- Single-molecule analysis offers detailed insights into biological processes.
Purpose of the Study:
- To integrate Rolling Circle Amplification (RCA) with solid-state nanopore sensing.
- To monitor RCA amplification kinetics and structural changes at the single-molecule level.
- To utilize miR-21 as a model biomarker for label-free RCA analysis.
Main Methods:
- Single-molecule monitoring of RCA using solid-state nanopore sensing.
- Analysis of DNA translocation dwell time and current blockage.
- Validation using Urea-PAGE, atomic force microscopy (AFM), and Mfold secondary structure prediction.
- Development of a custom signal extraction application for event classification.
Main Results:
- Demonstrated time-dependent increases in translocation dwell time and current blockage during RCA.
- Observed the generation of longer and more complex DNA concatemers.
- Confirmed structural evolution towards more stable configurations using AFM and Mfold.
- Achieved reproducible event classification and visualization with the custom application.
Conclusions:
- The integrated RCA and nanopore sensing platform provides novel insights into RCA amplification dynamics.
- This label-free approach enables structure-resolved analysis of nucleic acid amplification.
- Highlights the potential of nanopore sensing for developing advanced diagnostic tools.
