A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA interstrand crosslinks (ICLs) are critical DNA lesions that impede DNA replication and transcription.
• ICL-inducing agents are utilized in cancer therapeutics, necessitating effective methods for studying ICL formation and repair.
• Existing methods for detecting psoralen-induced ICLs often use large reporter groups that can interfere with biological activity.

Purpose of the Study:

• To develop a minimally-modified psoralen probe for efficient and accurate detection of DNA interstrand crosslinks (ICLs).
• To establish a click chemistry-based post-labeling strategy for visualizing and quantifying ICLs in cellular environments.
• To validate the utility of the new probe and method in studying cellular ICL repair pathways.

Main Methods:

• Synthesis of 8-propargyloxypsoralen (8-POP), a psoralen probe with a small alkyne handle.
• UVA activation of 8-POP in vitro and in cells to generate ICLs.
• Copper-catalyzed click chemistry for post-labeling of 8-POP-DNA lesions with azide-tagged fluorescent reporters.
• Modified alkaline comet assay, fluorescence microscopy, and flow cytometry for lesion detection and quantitation.
• Treatment with DNA repair inhibitors to assess lesion removal rates.

Main Results:

• UVA-activated 8-POP successfully generated ICLs in cellular systems.
• Click-mediated ligation enabled in situ detection and quantitation of 8-POP-DNA lesions using fluorescent reporters.
• The removal of 8-POP-DNA lesions was significantly reduced in the presence of DNA repair inhibitors, confirming 8-POP's suitability for repair studies.
• The post-labeling strategy proved to be inexpensive, rapid, and modular.

Conclusions:

• 8-propargyloxypsoralen (8-POP) serves as an effective probe for generating and detecting DNA interstrand crosslinks (ICLs) in cells.
• The click chemistry-based post-labeling approach provides a versatile and sensitive method for studying ICLs and DNA repair.
• This methodology offers a valuable tool for advancing research in DNA repair and the development of ICL-targeting therapeutics.