Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP₆‑Binding Proteins

Inositol hexakisphosphate (IP₆) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP₆ in critical cellular functions, few IP₆-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP₆ (IP₆-biotin), which has biotin attached at position 2 of the <i>myo</i>-inositol ring via an aminohexyl linker. Like natural IP₆, IP₆-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) <i>in vitro</i>. The Ku protein is a required NHEJ factor that has been shown to bind IP₆. We found that IP₆-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP₆-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP₆-biotin. IP₆-biotin was used to tether streptavidin to Ku, which inhibited NHEJ <i>in vitro</i>. These proof-of-concept experiments suggest that molecules like IP₆-biotin might be used to molecularly target biologically important proteins that bind IP₆. IP₆-biotin affinity capture experiments show that numerous proteins specifically bind IP₆-biotin, including casein kinase 2, which is known to bind IP₆, and nucleolin. Protein binding to IP₆-biotin is selective, as IP₃, IP₄, and IP₅ did not compete for binding of proteins to IP₆-biotin. Our results document IP₆-biotin as a useful tool for investigating the role of IP₆ in biological systems