title: Selenium-assisted nucleic acid crystallography: use of phosphoroselenoates for MAD phasing of a DNA structure.
abstract: The combination of synchrotron radiation and a variety of atoms or ions (either covalently attached to the biomolecule prior to crystallization or soaked into crystals) that serve as anomalous scatterers constitutes a powerful tool in the X-ray crystallographer's repertoire of structure determination techniques. Phosphoroselenoates in which one of the nonbridging phosphate oxygens in the backbone is replaced by selenium offer a simplified means for introducing an anomalous scatterer into oligonucleotides by conventional solid-phase synthesis. Unlike other methods that are used to derivatize DNA or RNA by covalent attachment of a heavy atom (i.e., bromine at the C5 position of pyrimidines), tedious synthesis of specialized nucleosides is not required. Introduction of selenium is readily accomplished in solid-phase oligonucleotide synthesis by replacing the standard oxidation agent with a solution of potassium selenocyanide. This results in a diastereomeric mixture of phosphoroselenoates that can be separated by strong anion-exchange HPLC. As a test case, all 10 DNA hexamers of the sequence CGCGCG containing a single phosphoroselenoate linkage (PSe) were prepared. Crystals were grown for a subset of them, and the structure of [d(C(PSe)GCGCG)](2) was determined by the multiwavelength anomalous dispersion technique and refined to 1.1 A resolution.
authors.affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Wilds
authors.forename: Rekha
authors.initials: R
authors.lastname: Pattanayek
authors.forename: Chongle
authors.initials: C
authors.lastname: Pan
authors.forename: Zdzislaw
authors.initials: Z
authors.lastname: Wawrzak
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D016566:Organoselenium Compounds / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D009841:Oligonucleotides; D016566:Organoselenium Compounds; D004247:DNA