Real-time monitoring and effector screening of APE1 based on rGO assisted DNA nanoprobe
Abstract:
Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.
Insights
Researchers developed a sensitive DNA nanoprobe to monitor human apurinic/pyrimidine endonuclease 1 (APE1) activity in cells. This method aids in screening APE1 inhibitors for cancer diagnosis and therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Human apurinic/pyrimidine endonuclease 1 (APE1) is crucial in tumor development and prognosis.
- APE1 overexpression and localization impact cancer progression, making it a therapeutic target.
- Intracellular APE1 activity detection is vital for cancer diagnosis, therapy, and inhibitor discovery.
Purpose of the Study:
- To develop a novel DNA-based nanoprobe for monitoring intracellular APE1 activity.
- To establish a sensitive and specific method for detecting APE1 enzyme activity.
- To utilize the developed system for screening APE1 inhibitors and real-time intracellular monitoring.
Main Methods:
- A DNA-based nanoprobe system was designed, comprising a single fluorophore-labeled hairpin probe and reduced graphene oxide (rGO).
- In vitro characterization determined the detection range (0.02–2 U/mL) and limit of detection (0.02 U/mL).
- Intracellular fluorescence imaging was employed for inhibitor screening and real-time monitoring of APE1 activity in A549 cells.
Main Results:
- The DNA nanoprobe demonstrated a linear response within the tested concentration range, with a low limit of detection.
- The method exhibited high specificity, enabling successful screening of APE1 inhibitors.
- Panaxytriol, identified from traditional Chinese medicine, was screened as an effective APE1 inhibitor, with its activity monitored in real-time.
Conclusions:
- The developed DNA-based nanoprobe offers a sensitive and specific platform for detecting intracellular APE1 activity.
- This technology facilitates the screening of APE1 inhibitors from natural sources like TCM.
- The APE1 detection system holds promise for advancing cancer diagnosis and therapeutic strategies.
