High-Resolution Mapping of Amino Acid Residues in DNA–Protein Cross-Links Enabled by Ribonucleotide-Containing DNA

DNA–protein cross-links have broad applications in mapping DNA–protein interactions and provide structural insights into macromolecular structures. However, high-resolution mapping of DNA-interacting amino acid residues with tandem mass spectrometry remains challenging due to difficulties in sample preparation and data analysis. Herein, we developed a method for identifying cross-linking amino residues in DNA–protein cross-links at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid–peptide cross-links under our optimized ribonucleotide cleavage conditions. The structurally defined oligonucleotide–peptide heteroconjugates improved ionization, reduced the database search space, and facilitated the identification of cross-linking residues in peptides. We applied the workflow to identifying abasic (AP) site-interacting residues in human mitochondrial transcription factor A (TFAM)-DNA cross-links. With sub-nmol sample input, we obtained high-quality fragmentation spectra for nucleic acid–peptide cross-links and identified 14 cross-linked lysine residues with the home-built AP_CrosslinkFinder program. Semi-quantification based on integrated peak areas revealed that K186 of TFAM is the major cross-linking residue, consistent with K186 being the closest (to the AP modification) lysine residue in solved TFAM:DNA crystal structures. Additional cross-linking lysine residues (K69, K76, K136, K154) support the dynamic characteristics of TFAM:DNA complexes. Overall, our combined workflow using ribonucleotide as a chemically cleavable DNA modification together with optimized sample preparation and data analysis offers a simple yet powerful approach for mapping cross-linking sites in DNA–protein cross-links. The method is amendable to other chemical or photo-cross-linking systems and can be extended to complex biological samples.