Ku protein as the main cellular target of cell-surface-bound circulating DNA

Objective: An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects. Materials and methods: The binding of [32P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane–cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry. Results and conclusions: Both ss- and ds-ODNs form strong ODN–protein complexes with similar electrophoretic mobilities after incubation with the MC...