A DNA/RNA hybrid fluorescent probe for high-throughput quantification of the activity of human apurinic/apyrimidinic endonuclease 1 in subcellular extracts

Peng Lu; Xiangjian Cao; Jinghui Zheng; Chenxv Zhu; Ruilan Zhang; Ying Sun; Ziyu Yang; Ziyu Tang; Jiayu Wang; Meiping Zhao

Biosensors and Bioelectronics: X (Sep 2023)

A DNA/RNA hybrid fluorescent probe for high-throughput quantification of the activity of human apurinic/apyrimidinic endonuclease 1 in subcellular extracts

Peng Lu,
Xiangjian Cao,
Jinghui Zheng,
Chenxv Zhu,
Ruilan Zhang,
Ying Sun,
Ziyu Yang,
Ziyu Tang,
Jiayu Wang,
Meiping Zhao

Affiliations

Peng Lu: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Xiangjian Cao: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Jinghui Zheng: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Chenxv Zhu: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Ruilan Zhang: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Ying Sun: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Ziyu Yang: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Ziyu Tang: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Jiayu Wang: Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
Meiping Zhao: Corresponding author.; Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China

Journal volume & issue: Vol. 14
p. 100329

Abstract

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As a multifunctional DNA repair enzyme, human apurinic/apyrimidinic endonuclease 1 (APE1) plays a pivotal role in many cellular processes. In recent years, it has been recognized as a potential prognostic biomarker and a therapeutic target for many cancers and other diseases. In this work, we report a DNA/RNA hybrid-based fluorescent probe which allows rapid and high-throughput detection of APE1 in the nuclear, cytoplasmic, and mitochondrial extracts of different types of cultured cells without any purification steps. As a noncanonical substrate, the DNA/RNA hybrid probe possesses inherent specificity toward APE1 without the need of additional chemical modification. A substantial acceleration effect of the 3′-flanking mismatches enabled rapid digestion of the abasic sites in the DNA/RNA hybrid by APE1 at a comparable rate to those in natural double-stranded DNA. The hybrid probe showed adequately high sensitivity to the target enzyme (linear working range: 0.02–1.0 U/mL; detection limit: 0.01 U/mL) and superior resistance to interference from cellular proteins. The mechanism for the 3′-mismatch enhancement was also clarified via kinetic study and single-molecule fluorescence analysis. By using the probe, significantly varied subcellular distribution of APE1 in different cells were disclosed, showing the importance of quantitative measurement of APE1 activity in subcellular compartments for drug screening and evaluation of the treatment efficacy. The method was simple and robust, which offered an easily accessible molecular tool for high-throughput quantification of the subcellular APE1 activity. It may also facilitate the development of effective anticancer drugs.

Published in Biosensors and Bioelectronics: X

ISSN: 2590-1370 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Technology: Chemical technology: Biotechnology
Website: https://www.journals.elsevier.com/biosensors-and-bioelectronics-x

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