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Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons
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Allosteric Genetically Encoded Dual-Color Fluorogenic RNA Biosensor Simultaneously Illuminates miRNA and Its

Wen-Jing Liu1, Hai-Juan Li1, Zichen Jiao2

  • 1School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China.

Analytical Chemistry
|October 17, 2025
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Summary

We developed a novel dual-color RNA biosensor for sensitive, label-free detection of microRNAs (miRNAs) and pre-miRNAs. This tool enables precise monitoring in cells and tissues, aiding in early disease diagnosis.

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Area of Science:

  • Biomolecular Engineering
  • Molecular Diagnostics
  • RNA Biology

Background:

  • MicroRNAs (miRNAs) and their precursors (pre-miRNAs) are key biomarkers in physiological and pathological processes.
  • Understanding their relationship is crucial for disease mechanism insights and early diagnosis.
  • Existing detection methods often require complex procedures or labels.

Purpose of the Study:

  • To develop a simple, sensitive, and label-free biosensor for simultaneous detection of miRNA and pre-miRNA.
  • To enable monitoring of these nucleic acids in complex biological samples like cells and tissues.
  • To establish a versatile platform for nucleic acid detection.

Main Methods:

  • Construction of a dual-color fluorogenic RNA biosensor utilizing a hairpin allosteric-driven transcription circuit.
  • Design of a multifunctional hairpin probe for target recognition, transcription amplification, and signal transduction.
  • Utilizing aptamer-fluorophore complexes for signal generation upon target binding.

Main Results:

  • The biosensor achieved isothermal, one-step, homogeneous detection of miR-155 and pre-miR-155 with attomolar sensitivity.
  • Accurate quantification and single-cell level analysis of miRNA and pre-miRNA were demonstrated.
  • Differential expression profiles between breast cancer and healthy tissues were successfully discriminated.

Conclusions:

  • The developed RNA biosensor offers a powerful tool for sensitive and specific detection of miRNAs and pre-miRNAs.
  • This platform provides a new paradigm for precise nucleic acid diagnostics and disease research.
  • The biosensor's scalability allows for adaptation to detect various other nucleic acid targets.