Directed cleavage of RNA with protein-tethered EDTA-Fe.
There are several methods for locating the RNA site where a protein binds. One of the less common methods is directed cleavage of the RNA by an EDTA-Fe reagent tethered to the protein. The reaction of the EDTA-Fe(III) with ascorbate or hydrogen peroxide produces reactive oxygen species, such as hydroxyl radicals, localized within a 10-A radius of the iron center. The reactive oxygen species will attack the ribose or deoxyribose of nucleic acids as well as proximal polypeptide backbones. One EDTA-Fe reagent, (EDTA-2-aminoethyl)-2-pyridyl disulfide complexed to iron (EPD-Fe), has been tethered to several proteins through a disulfide linkage to engineered cysteine thiols and used to cleave DNA, proteins, and RNA. A second tethered EDTA-Fe reagent, 1-(p-bromoacetamidobenzyl)-EDTA-Fe, or BABE, has also been used to cleave RNA. Here we describe the issues involved in using these reagents with any RNA binding protein.
Hall
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Fox
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D002614:Chelating Agents / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology*; D008967:Molecular Biology / Q000379:methods*; D009942:Organometallic Compounds / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*
D002614:Chelating Agents; D009942:Organometallic Compounds; D012313:RNA; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D007501:Iron