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Updated: Jan 2, 2026
MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as A Novel Detection and Quantification Method
Published on: October 7, 2025
G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA
Hong-Xin Jiang1, Zhen-Zhen Liang2, Yan-Hong Ma2
1State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, PR China; Collaborative Innovation Centre of Chemical Science and Engineering (Tianjin), Tianjin, 300071, PR China; Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, Nankai University, Tianjin, 300071, PR China.
This study introduces a novel real-time monitoring strategy for rolling circular amplification (RCA) using thioflavin T (ThT) fluorescence. This method enables sensitive and specific detection of DNA amplification, advancing biosensing applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Analytical Chemistry
Background:
- Real-time PCR enables quantitative DNA analysis.
- Rolling circular amplification (RCA) is a versatile isothermal DNA amplification technique.
- A simple, sensitive, and specific real-time monitoring strategy for RCA is needed to enhance its utility.
Purpose of the Study:
- To develop a real-time monitoring strategy for rolling circular amplification (RCA).
- To utilize the specific fluorescence of thioflavin T (ThT) in response to G-quadruplexes formed by RCA products.
- To adapt this strategy for use in commercially available real-time PCR instruments for biosensing applications.
Main Methods:
- Employed thioflavin T (ThT) as a fluorescent probe that binds to G-quadruplex structures formed during RCA.
- Applied the ThT-based fluorescence detection to both linear and exponential RCA amplification.
- Integrated the method with standard real-time PCR instruments for high-throughput analysis.
Main Results:
- Demonstrated successful real-time monitoring of RCA using ThT fluorescence.
- Achieved broad linear ranges (8 orders of magnitude) for microRNA quantitation with a detection limit of 4 aM.
- Successfully applied the RCA-based sensing platform for microRNA analysis in human lung cancer cells.
Conclusions:
- Developed a novel, sensitive, and specific real-time monitoring method for RCA using G-quadruplex-ThT interactions.
- The technique is compatible with standard real-time PCR instruments, facilitating high-throughput biosensing.
- This approach holds potential for broader application in isothermal DNA amplification techniques and diagnostics.
