On-bead fluorescent DNA nanoprobes to analyze base excision repair activities

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA integrity is crucial and constantly challenged by endogenous and exogenous agents, leading to mutations associated with diseases like cancer.
• Organisms possess sophisticated DNA repair mechanisms, including base excision repair (BER), to counteract DNA damage.
• Accurate detection of BER enzyme activity is vital for understanding DNA repair pathways and developing therapeutic interventions.

Purpose of the Study:

• To develop and validate an original, sensitive biosensor assay for detecting DNA repair enzyme activity within the base excision repair (BER) pathway.
• To characterize the assay's performance using specific BER enzymes, alkyl-adenine glycosylase (AAG) and human AP-endonuclease (APE1).
• To demonstrate the assay's utility in screening for inhibitors of DNA repair enzymes.

Main Methods:

• Development of a biosensor utilizing fluorescent hairpin-shaped nucleic acid probes immobilized on magnetic beads.
• Incorporation of specific DNA lesions (hypoxanthine for AAG, tetrahydrofuran analog for APE1) into the probes.
• Detection of enzymatic repair activity via fluorescence measurement of cleaved probes in the supernatant.

Main Results:

• The assay successfully detected the activity of AAG and APE1 enzymes in a fast, specific, and quantitative manner.
• A low detection limit was achieved, with 1 μg mL⁻¹ for APE1 and 50 μg mL⁻¹ for AAG using HeLa nuclear extracts.
• The assay requires minimal probe amounts (1 pmol per test), indicating high efficiency and sensitivity.

Conclusions:

• The developed on-bead fluorescent biosensor assay provides a robust method for measuring DNA repair enzyme activities.
• This assay is suitable for analyzing purified enzymes and cell-free extracts, offering a sensitive and quantitative approach.
• The assay's design makes it a valuable tool for screening potential inhibitors of DNA repair enzymes, relevant for cancer therapy and genetic disease research.