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A Primer-Regulated Rolling Circle Amplification (RCA) for Logic-Controlled Multiplexed Enzyme Analysis.

Shuiqin Chai¹, Wanlin Sun¹, Xin Hou¹

¹College of Chemistry and Chemical Engineering, Chongqing University of Science and Technology, Chongqing 401331, PR China.

ACS Applied Bio Materials

|February 21, 2025

Summary

A new primer-regulated rolling circle amplification (RCA) method detects DNA-related enzymes. This ultrasensitive assay unlocks enzyme activity for biomarker detection and disease diagnosis.

Keywords:

DNA-related enzymes logic operations modular design multiple analyses primer-regulated RCA

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Area of Science:

Biochemistry
Molecular Biology
Enzyme kinetics

Background:

DNA-related enzymes are crucial biomarkers for disease diagnosis.
Developing ultrasensitive detection methods for these enzymes is essential for clinical applications.

Purpose of the Study:

To develop a novel, robust, and ultrasensitive strategy for detecting DNA-related enzymes.
To create a universal tool for single or multiple enzyme analyses using a logic-controlled approach.

Main Methods:

A primer-regulated rolling circle amplification (RCA) strategy was designed.
Primers were modified with 3'-inverted dT to prevent premature amplification (locked state).
Enzyme recognition substrates were embedded in primers, releasing 3'-inverted dT upon target interaction (unlocked state) to initiate RCA.

Main Results:

The strategy successfully initiated RCA upon specific enzyme recognition and cleavage.
The method demonstrated programmability for detecting single base sites or specific sequences.
Logic-controlled operations (YES, OR, AND, AND-OR) enabled versatile single and multiplexed enzyme detection.

Conclusions:

The proposed primer-regulated RCA strategy offers a universal and modular tool for diverse DNA-related enzyme detection.
This method provides a robust and ultrasensitive approach for biomarker analysis and clinical diagnosis.
The logic-controlled system allows for convenient and adaptable enzyme assays.