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Updated: Sep 13, 2025
Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
Published on: June 8, 2018
A cell-permeable fluorescent probe for imaging of DNA repair protein ALKBH2
Unnikrishnan P Shaji1, Rashmi Ranjan Khatua2, Sourbh Rankawat3
1DNA Repair Research Lab, Department of Biotechnology, IIT Hyderabad, Kandi, Telangana, 502284, India.
Abstract:
ALKBH2 is a key DNA repair enzyme that removes various methyl-adducts, including N1-methyl-adenine and N3-methyl-cytosine, from genomic DNA. ALKBH2 overexpression is observed in many cancer and leads to temozolomide resistance in glioblastoma cell lines. The growing significance of ALKBH2 as a promising therapeutic target in cancer has highlighted the need for suitable chemical tools to enable its detailed study and functional characterization. To address this gap, we developed a fluorescent probe for rapid and selective detection of ALKBH2 in live cancer cells. Building on our previously reported finding that the HIV protease inhibitor ritonavir selectively binds and inhibits ALKBH2, we synthesized a BODIPY-labeled ritonavir (rit-BD) as an imaging agent. Rit-BD showed peak absorption (Amax) and emission (Emax) to be 576 nm and 586 nm, respectively. Our results from the binding experiment demonstrate that rit-BD binds to ALKBH2 in vitro with micromolar affinity. The limit of detection (LOD) for ALKBH2 with Rit-BD was estimated to be 115 ng/ml (4.7 nM) with a linear range of 0.5-330 μg/ml (14 nM-10 μM). Furthermore, In vitro and in cellulo experiments and live-cell imaging confirmed that rit-BD could be used to label ALKBH2 in the living cell nucleus. Our findings establish rit-BD as a unique dual-purpose agent for visualizing ALKBH2 dynamics and inhibiting DNA repair activity in cancer cells.
Insights
Researchers developed a novel fluorescent probe, ritonavir-BODIPY (rit-BD), for detecting the DNA repair enzyme ALKBH2 in live cancer cells. This probe aids in studying ALKBH2
Area of Science:
- Biochemistry and Molecular Biology
- Chemical Biology
- Cancer Research
Background:
- ALKBH2 is a crucial DNA repair enzyme implicated in cancer development and temozolomide resistance.
- Overexpression of ALKBH2 in various cancers necessitates effective tools for its study and therapeutic targeting.
- Existing methods for ALKBH2 detection and functional analysis are limited, creating a need for novel chemical probes.
Purpose of the Study:
- To develop a fluorescent probe for rapid and selective detection of ALKBH2 in live cancer cells.
- To create a chemical tool for visualizing ALKBH2 dynamics and inhibiting its DNA repair activity.
- To leverage the known ALKBH2 inhibitory properties of ritonavir for probe development.
Main Methods:
- Synthesis of a BODIPY-labeled ritonavir derivative (rit-BD) based on ritonavir's selective binding to ALKBH2.
- Characterization of rit-BD's spectral properties (absorption and emission maxima).
- In vitro binding assays to determine rit-BD's affinity for ALKBH2.
- Live-cell imaging experiments to visualize ALKBH2 localization and dynamics within cancer cells.
Main Results:
- Rit-BD exhibited peak absorption at 576 nm and emission at 586 nm.
- Rit-BD demonstrated micromolar binding affinity for ALKBH2 in vitro.
- The probe achieved a limit of detection of 115 ng/ml (4.7 nM) for ALKBH2.
- Live-cell imaging confirmed rit-BD's ability to label ALKBH2 within the nucleus of living cancer cells.
Conclusions:
- Rit-BD is a novel fluorescent probe effective for detecting and visualizing ALKBH2 in live cancer cells.
- The probe serves a dual purpose: imaging ALKBH2 dynamics and potentially inhibiting its DNA repair function.
- Rit-BD represents a valuable chemical tool for advancing research into ALKBH2's role in cancer and DNA repair.
