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Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...
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One-Pot Rlock-Mediated CRISPR/Cas12a-Driven RCA Cycle for Rapid and High-Sensitive APE1 Detection.

Junzhuo Shan1, Yixiao Sheng1, Lun Luo1

  • 1School of Biology and Biological Engineering, South China University of Technology, Guangzhou, Guangdong Province 510006, China.

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|August 15, 2025
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Summary
This summary is machine-generated.

A new Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE) assay enables rapid, sensitive point-of-care testing for Apurinic/apyrimidinic endonuclease 1 (APE1). This innovation aids early cancer diagnosis with minimal equipment and cost.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Apurinic/apyrimidinic endonuclease 1 (APE1) is crucial in DNA repair and redox regulation, often overexpressed in tumors.
  • Early cancer diagnosis necessitates sensitive point-of-care testing (POCT) for APE1.
  • Existing Rolling Circle Amplification (RCA) methods are rarely applied to APE1 detection.

Purpose of the Study:

  • To develop a novel POCT strategy for APE1 detection.
  • To create a versatile platform for converting RCA-based assays into APE1-responsive systems.
  • To integrate CRISPR/Cas12a and RCA for a one-pot APE1 detection system.

Main Methods:

  • Introduction of the RCA-Lock (Rlock) conversion platform.
  • Development of the Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE) assay.
  • Integration of CRISPR/Cas12a and RCA in a one-pot system for APE1 detection.

Main Results:

  • The RCRE assay achieved a limit of detection of 8.86 × 10-4 U/mL.
  • Detection was accomplished within 30 minutes.
  • The assay requires minimal equipment, is low-cost, and involves simple procedures.

Conclusions:

  • The Rlock strategy offers a transformative advance in APE1 diagnostics.
  • The RCRE assay provides a programmable, nucleic acid-based biosensor for APE1.
  • This technology broadens options for early cancer detection and clinical translation.