Hydroxyl radical footprinting

Abstract Hydroxyl radicals have now been used for more than 15years to probe the structure of various macromolecules. They have also been widely used to footprint DNA-protein and RNA-protein complexes (1, 2). Hydroxyl radicals are w1y small, comparable in size to a water molecule, are most commonly generated from FeEDTA in solution by an innocuous and inexpensive procedure (see Protocols 2 and 3) <md are capable of cutting the DNA or RNA backbone at eve1y accessible nucleotide without sequence specificity (3-5). A footprinting reagent in which the .EDTA moiety is coupled to an intercalator can also be used to generate hydroxyl radicals (6). This reactant. methidium-propyl Fe-EDTA, binds to some preferred positions along the DNA, and consequently the pattern of DNA backbone cleavage is much less random than with Fee-EDTA alone.