title: Site-specific platinum(II) cross-linking in a ribozyme active site.
abstract: The function of RNA depends on its ability to adopt complex and dynamic structures, and the incorporation of site-specific cross-linking probes is a powerful method for providing distance constraints that are valuable in RNA structural biology. Here we describe a new RNA-RNA cross-linking strategy based on Pt(II) targeting of specific phosphorothioate substitutions. In this strategy cis-diammine Pt(II) complexes are kinetically recruited and anchored to a phosphorothioate substitution embedded within a structured RNA. Substitution of the remaining exchangeable Pt(II) ligand with a nucleophile supplied by a nearby RNA nucleobase results in metal-mediated cross-links that are stable during isolation. This type of cross-linking strategy was explored within the catalytic core of the Hammerhead ribozyme (HHRz). When a phosphorothioate substitution is installed at the scissile bond normally cleaved by the HHRz, Pt(II) cross-linking takes place to nucleotides G8 and G10 in the ribozyme active site. Both of these positions are predicted to be within ~8 Å of a phosphorothioate-bound Pt(II) metal center. Cross-linking depends on Mg(2+) ion concentration, reaching yields as high as 30%, with rates that indicate cation competition within the RNA three-helix junction. Cross-linking efficiency depends on accurate formation of the HHRz tertiary structure, and cross-links are not observed for RNA helices. Combined, these results show promise for using kinetically inert Pt(II) complexes as new site-specific cross-linking tools for exploring RNA structure and dynamics.
authors: Department of Chemistry and Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
authors: Erich G
authors: EG
authors: Chapman
authors: Victoria J
authors: VJ
authors: DeRose
mesh_terms: D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009944:Organoplatinum Compounds / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D009944:Organoplatinum Compounds; D010710:Phosphates; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; C083120:phosphorodithioic acid