Label-free and highly sensitive APE1 detection based on rolling circle amplification combined with G-quadruplex

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Apurinic/apyrimidinic endonuclease 1 (APE1) activity is crucial for DNA repair and implicated in various diseases.
• Existing methods for APE1 detection often suffer from low sensitivity, complex labeling, or narrow linear ranges.
• Highly sensitive and label-free detection methods are needed for accurate APE1 assessment in biological samples.

Purpose of the Study:

• To develop a novel, highly sensitive, and label-free fluorescence detection method for apurinic/apyrimidinic endonuclease 1 (APE1) activity.
• To leverage rolling circle amplification (RCA) and G-quadruplex (G4) structures for enhanced signal generation.
• To validate the method's performance in biological matrices like serum.

Main Methods:

• A hairpin probe containing an AP site is cleaved by APE1, releasing a primer to initiate RCA.
• Rolling circle amplification generates long DNA amplicons with repeating sequences.
• The amplicon forms G-quadruplex structures that bind Thioflavin T (ThT), producing a detectable fluorescence signal.

Main Results:

• The developed RCA-G4 method achieved a low detection limit of 1.52 × 10⁻⁶ U/mL for APE1 activity.
• A wide linear range from 2 × 10⁻⁶ to 10 U/mL was established for APE1 quantification.
• The method demonstrated successful application in serum samples with high recovery rates (96.3%–105.7%).

Conclusions:

• The RCA-G4 method provides a highly sensitive and label-free approach for detecting APE1 activity.
• This technique overcomes limitations of previous methods, offering improved sensitivity and simplified procedures.
• The developed assay holds promise for advancing APE1-related disease research and drug development.