Fast photochemical oxidation of nucleic acids maps protein- DNA interactions at sub-nucleotide resolution

Le résumé fourni par la source

Transcription factor (TF) binding to DNA reshapes its backbone accessibility and modulate gene expression. However, protein–DNA interactions remain difficult to characterize with high resolution at the residue level. Here, we introduce Fast Photochemical Oxidation of Nucleic acids (FPON), a laser-driven hydroxyl-radical footprinting workflow coupled to liquid chromatography–high-resolution mass spectrometry (LC-MS) analysis. FPON quantitatively maps DNA accessibility and damage at sub-nucleotide resolution, revealing structural and interaction patterns induced by TF binding. Using the Forkhead box O4 DNA‑binding domain (FOXO4-DBD) bound to a double-stranded insulin response element (IRE), FPON generated structurally informative DNA fragments resolved and assigned by LC-MSn in a single experiment. Quantitative analysis identified site-specific protection patterns within the FOXO4 recognition motif. In the absence of a high-resolution structure, these site-specific protection patterns guided the construction of an in silico FOXO4–IRE complex. Complementary molecular dynamics simulations revealed a strong agreement between experimentally observed cleavage suppression and reduced solvent accessibility of DNA backbone hydrogens. Furthermore, FPON resolved multiple chemically distinct cleavage products otherwise indistinguishable in conventional gel-based footprinting approaches. Together, these results establish FPON as an unbiased, high-resolution strategy for mapping protein-DNA contacts readily applicable to more complex chromatin systems and dynamic transcriptional processes.

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