DNA-protein cross-links (DPCs) are super-bulky DNA adducts induced by common chemotherapeutic agents, reactive oxygen species, and aldehydes, and also formed endogenously as part of epigenetic regulation. Despite their presence in most cells and tissues, the biological effects of DPCs are poorly understood due to the difficulty of constructing site-specific DNA-protein conjugates. In the present work, a new approach of conjugating proteins to DNA using oxime ligation was used to generate model DPCs structurally analogous to lesions formed in cells. In our approach, proteins and peptides containing an unnatural oxy-Lys amino acid were cross-linked to DNA strands functionalized with 5-formyl-dC or 7-(2-oxoethyl)-7-deaza-dG residues using oxime ligation. The conjugation reaction was site-specific with respect to both protein and DNA, provided excellent reaction yields, and formed stable DPCs amenable to biological evaluation.
Bibliographical noteFunding Information:
We thank Dr Guru S. Madugundu and Yingchun Zhao for their help with MS analysis of DPCs, Dr M. Rashidian and H. Ploegh for providing recombinant Sortase A and pHEN6, and Robert Carlson (UMN Masonic Cancer Center) for help with editing and the graphics for the manuscript. This research was funded by NIH grants ES-023350 and GM-084152.
© 2018 The Royal Society of Chemistry.