Advantages of Hydroxyl Radical Footprinting

The hydroxyl radical footprinting technique has several advantages (Shafer etai, 1989). Nucleic acid strand scission is mediated by hydroxyl radicals (Pogozelski et al, 1995, and references therein) generated by the reduction of hydrogen peroxide (H202) or dioxygen (02) by iron(II) (Bull et al., 1983; Tullius and Dombroski, 1986). Since all sugar moieties accessible to solvent (Hertzberg and Dervan, 1984; Wu et al., 1983; Wu and Kozarich, 1985) are the target of attack by these small, diffusible radicals (Pogozelski et al., 1995, and references therein), minimal specificity (Shafer et al., 1989) for either nucleotide sequence (Henner et al., 1982; Hertzberg and Dervan, 1984; Tullius and Dombroski, 1985) or secondary structure (Celander and Cech, 1990) is evident. Due to these properties, the hydroxyl radical is an excellent probe for a high-resolution analysis of backbone regions protected by specific nucleic acid-protein interactions (Tullius and Dombroski, 1986; Wang and Padgett, 1989). In addition, the Fe(II)-EDTA complex used in the reactions is negatively charged and therefore unlikely to either bind or intercalate into nucleic acids, thereby potentially changing their conformation (Shafer et al., 1989). Finally, Fe(II)—EDTA-catalyzed cleavage reactions can occur under a wide range of buffer and reaction conditions (Celander and Cech, 1990; Tullius et al., 1987; Wang and Padgett, 1989), thus making the hydroxyl radical footprinting method amenable to varied experimental systems and protocols.

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