Alkene-tetrazine ligation for imaging cellular DNA

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Cellular DNA synthesis is crucial for cell replication and requires accurate monitoring.
• Current methods for visualizing DNA synthesis, like using 5-ethynyl-2 ahydro-deoxyuridine (EdU), rely on azide-alkyne click chemistry.
• There is a need for alternative bioorthogonal labeling strategies with reduced genotoxicity and orthogonal reactivity.

Purpose of the Study:

• To introduce 5-Vinyl-2 ahydro-deoxyuridine (VdU) as a novel metabolic probe for visualizing DNA synthesis.
• To evaluate the incorporation, genotoxicity, and ligation efficiency of VdU in replicating cells.
• To establish VdU-tetrazine ligation as a bioorthogonal alternative to EdU's click reaction for nucleic acid labeling.

Main Methods:

• VdU was synthesized and tested for its incorporation into cellular DNA by endogenous enzymes.
• The inverse electron demand Diels-Alder reaction between VdU and a fluorescent tetrazine was optimized for labeling.
• The chemical orthogonality of the VdU-tetrazine reaction was compared to the EdU-azide-alkyne click reaction.

Main Results:

• VdU is efficiently incorporated into the genomes of replicating cells.
• VdU exhibits reduced genotoxicity compared to EdU.
• The VdU-tetrazine ligation reaction is rapid (k≈0.02 M⁻¹ s⁻¹) and bioorthogonal to azide-alkyne click chemistry.
• This enables time-resolved and multicolor labeling of DNA synthesis.

Conclusions:

• VdU is the first metabolic probe for DNA synthesis visualized via inverse electron demand Diels-Alder reaction.
• VdU offers a safer and effective bioorthogonal labeling strategy for nucleic acids.
• Alkene-tetrazine ligation provides a valuable alternative to click chemistry for studying DNA synthesis in cells.