Directed cleavage of RNA with protein-tethered EDTA-Fe |
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Authors: | Hall K B Fox R O |
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Affiliation: | Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. |
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Abstract: | 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. |
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