Cross-linking cellular nucleic acids via a target-directing double click reagent

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Bioorthogonal ligation reactions are crucial for studying DNA metabolism, DNA-protein interactions, and developing therapeutics.
• Incorporating nucleoside analogs with bioorthogonal functional groups into chromatin is often limited by insufficient kinase phosphorylation and steric hindrance within DNA.

Purpose of the Study:

• To describe methods overcoming limitations in bioorthogonal functionalization of DNA.
• To enable efficient incorporation of azide groups into DNA and facilitate subsequent click reactions within chromatin.

Main Methods:

• Utilizing nucleotide monophosphate triesterers to bypass cellular nucleoside kinase activity for efficient azide incorporation into DNA.
• Employing a bioorthogonal intercalating reagent (cationic Sondheimer diyne) for steric access and modification of azide groups in folded chromatin.

Main Results:

• Efficient incorporation of azide groups into cellular DNA is achieved by bypassing endogenous kinase activity.
• The intercalating reagent enables tandem, strain-promoted cross-linking of azides, forming DNA-DNA interstrand crosslinks or DNA-fluorophore conjugates within native chromatin.

Conclusions:

• The described methods enhance the efficiency and accessibility of bioorthogonal reactions in DNA.
• These advancements offer new possibilities for DNA research, diagnostics, and therapeutic strategies.