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Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems
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Autonomous Exponential Amplification via Rolling Circle-DNAzyme Feedback Programming.

Jinhua Shang1, Mengdi Yu1, Yifei Wang1

  • 1College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.

Journal of the American Chemical Society
|November 26, 2025
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Summary
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This study introduces exponential rolling circle amplification (E-RCA), a novel nucleic acid detection method that uses self-regenerating primers for enhanced sensitivity. This DNA-based system offers programmable and autonomous molecular analysis for biological and clinical applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Biochemistry

Background:

  • Rolling circle amplification (RCA) is a sensitive isothermal nucleic acid detection method.
  • Traditional RCA has limitations in sensitivity and programmability due to linear kinetics and reliance on external primers.

Purpose of the Study:

  • To develop an exponential RCA (E-RCA) platform for enhanced nucleic acid detection.
  • To integrate primer regeneration and signal amplification into a single DNA-encoded system.

Main Methods:

  • Designed a circular DNA template with self-cleaving DNAzyme sequences for primer regeneration during RCA.
  • Utilized a DNA polymerase for amplification without external primers or protein enzymes.
  • Validated the E-RCA system in multiplexed intracellular microRNA imaging and breast cancer tissue profiling.

Main Results:

  • Achieved exponential signal amplification through a self-sustained (RCA ↔ DNAzyme) circuit.
  • Demonstrated high diagnostic accuracy (AUC = 0.914, specificity = 100%, sensitivity = 81.3%) in clinical samples.
  • Successfully performed multiplexed intracellular microRNA imaging and dual-marker profiling.

Conclusions:

  • The E-RCA platform provides sensitive, programmable, and autonomous molecular analysis.
  • This DNA-based strategy overcomes limitations of traditional RCA for advanced biological and clinical applications.