A DNA array based on clickable lesion-containing hairpin probes for multiplexed detection of base excision repair...

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• DNA damage from reactive species can lead to mutations, aging, and diseases like cancer.
• Accurate measurement of DNA repair activity is crucial for understanding these processes.
• Existing assays for DNA repair analysis can be complex and time-consuming.

Purpose of the Study:

• To develop a miniaturized, parallelized, on-surface DNA biosensor for analyzing base excision repair (BER) activities.
• To create a high-throughput method for detecting DNA N-glycosylase and AP-endonuclease activities.
• To evaluate the efficacy of BER inhibitors using this new platform.

Main Methods:

• Utilized "click chemistry" to immobilize fluorescent stem-loop DNA probes with specific lesions on a glass slide.
• Developed a miniaturized and parallelized on-surface DNA biosensor.
• Detected uracil N-glycosylase and AP-endonuclease activities in purified enzymes and cell extracts.

Main Results:

• Successfully detected uracil N-glycosylase and AP-endonuclease activities using the developed biosensor.
• Demonstrated the ability to analyze the inhibitory effects of Uracil glycosylase inhibitor (Ugi) and methoxyamine (MX) on BER pathways.
• Validated the biosensor's performance with both purified enzymes and complex cell extracts.

Conclusions:

• The new fluorescent DNA microarray platform offers an easy, rapid, and robust method for DNA repair analysis.
• This technology enables multiplexed detection of DNA N-glycosylase and AP-endonuclease activities.
• The biosensor is effective for evaluating the impact of BER inhibitors, advancing research in DNA repair and related diseases.