Comparison of rRNA Cleavage by Complementary 1,10-Phenanthroline-Cu(II)- and EDTA-Fe(II)-Derivatized Oligonucleotides

Abstract

The chemical nucleases 1,10-phenanthroline-Cu(II) and EDTA-Fe(II), have proven to be valuable tools for structural analysis of nucleic acids. Both have found applications in footprinting and directed proximity studies of DNA and RNA. Derivatives of each that provide for tethering to nucleic acid or protein are commercially available, allowing their widespread use for structural analysis of macromolecules. Although their applications are somewhat overlapping, differences in their cleavage mechanisms and chemical properties allow them to provide distinct and complementary structural information. The purpose of this study is to compare directly the cleavage patterns of tethered 1,10-phenanthroline-Cu(II) and EDTA-Fe(II) complexes within a similar experimental system. Here, the region surrounding nucleotide 1400 of 16S rRNA from Escherichia coli serves as a substrate for chemical cleavage directed by a derivatized complementary oligonucleotide. This region of rRNA is known to be involved in the decoding of mRNA during translation. The results of this study provide evidence in support of the mechanistic differences previously established for EDTA-Fe(II) and 1,10-phenathroline-Cu(II). The delocalized cleavage envelope produced by EDTA-Fe(II) cleavage suggests the involvement of a diffusible reactive species. On the other hand, rRNA cleavage induced by the tethered 1,10-phenanthroline-Cu(II) complex appears localized to the proximity of the chemical nuclease under normal conditions, although the production of an unknown diffusible species appears to occur during long reaction times.