Real-Time, Light-Activated, and Multiplexed Monitoring of Base Excision Repair in Living Cells Using Chimeric d/l-DNA...

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Base excision repair (BER) is crucial for DNA lesion repair.
• Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in BER and a disease biomarker.
• Existing APE1 probes are unsuitable for live-cell applications.

Purpose of the Study:

• To develop a versatile probe for intracellular APE1 detection and monitoring.
• To enable real-time and light-controlled analysis of BER in living cells.
• To facilitate the development of improved BER-targeted therapies.

Main Methods:

• Utilized bio-orthogonal properties of l-DNA and a chimeric d/l-DNA molecular beacon architecture.
• Developed a probe for intracellular detection and real-time monitoring of APE1 activity.
• Applied the probe for multiplexed imaging of APE1 and DNA glycosylase activities.

Main Results:

• The chimeric probe allows for simple, fast, and biostable monitoring of APE1 in living cell nuclei.
• Demonstrated the ability to distinguish cells by APE1 expression levels and monitor dynamic activity.
• Enabled simultaneous imaging of APE1 and DNA glycosylase, revealing insights into APE1 inhibitor efficacy.

Conclusions:

• The novel chimeric d/l-DNA beacon probe is a versatile tool for studying BER.
• This probe facilitates diverse intracellular applications and the development of BER-targeted therapies.
• The probe offers a new toolkit for researchers investigating DNA repair mechanisms and therapeutic strategies.