DNase I Footprinting of Small Molecule Binding Sites on DNA

Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.