title: Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.
abstract: Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E²⁸⁻²¹⁷ specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.
authors.forename: Sylwia Anna
authors.initials: SA
authors.lastname: Szczepaniak
authors.forename: Joanna
authors.initials: J
authors.lastname: Zuberek
authors.forename: Edward
authors.initials: E
authors.lastname: Darzynkiewicz
authors.forename: Joanna
authors.initials: J
authors.lastname: Kufel
authors.forename: Jacek
authors.initials: J
authors.lastname: Jemielity
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D039561:Eukaryotic Initiation Factor-4E / Q000737:chemistry; D006801:Humans; D051379:Mice; D011485:Protein Binding; D012314:RNA Cap Analogs / Q000737:chemistry*; D039381:RNA Cap-Binding Proteins / Q000032:analysis / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives / Q000138:chemical synthesis*
chemical_list: D039561:Eukaryotic Initiation Factor-4E; D012314:RNA Cap Analogs; D039381:RNA Cap-Binding Proteins; C509442:m7GpppA; D012685:Sepharose