title: Syntheses of RNAs with up to 100 nucleotides containing site-specific 2'-methylseleno labels for use in X-ray crystallography.
abstract: The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1,4-dimercapto-2,3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.
authors.affiliation: Institute of Organic Chemistry, Center for Molecular Biosciences (CMBI), Leopold-Franzens University, 6020 Innsbruck, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Höbartner
authors.forename: Renate
authors.initials: R
authors.lastname: Rieder
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreutz
authors.forename: Barbara
authors.initials: B
authors.lastname: Puffer
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.forename: Anna
authors.initials: A
authors.lastname: Polonskaia
authors.forename: Alexander
authors.initials: A
authors.lastname: Serganov
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
mesh_terms: D001483:Base Sequence; D002072:Butylene Glycols / Q000737:chemistry; D018360:Crystallography, X-Ray; D004112:Dimercaprol / Q000737:chemistry; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D009943:Organophosphorus Compounds / Q000138:chemical synthesis; D016566:Organoselenium Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D011687:Purines / Q000737:chemistry; D012313:RNA / Q000138:chemical synthesis*; D016337:RNA, Catalytic / Q000737:chemistry; D012263:Ribonucleosides / Q000737:chemistry
chemical_list: D002072:Butylene Glycols; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D016566:Organoselenium Compounds; D011687:Purines; D016337:RNA, Catalytic; D012263:Ribonucleosides; C434331:phosphoramidite; D004112:Dimercaprol; D012313:RNA; D008025:Ligases