Selenium-Assisted Nucleic Acid Crystallography: Use of DNA Phosphoroselenoates for MAD Phasing

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 determi...

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Bibliographic Details
Published inJournal of the American Chemical Society Vol. 124; no. 2002
Main Authors Wilds, C.J., Pattanayek, R., Pan, C., Wawrzak, Z., Egli, M.
Format Journal Article
LanguageEnglish
Published United States 08.03.2010
Subjects
ATOMS
BROMINE
CRYSTALLIZATION
CRYSTALLOGRAPHY
DNA
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
MIXTURES
NUCLEIC ACIDS
NUCLEOSIDES
OLIGONUCLEOTIDES
OXIDATION
PARTICLE ACCELERATORS
PHOSPHATES
POTASSIUM
PYRIMIDINES
RESOLUTION
RNA
SELENIUM
SYNCHROTRON RADIATION
SYNTHESIS
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Summary: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{sub PSe}GCGCG)]{sub 2} was determined by the multiwavelength anomalous dispersion technique and refined to 1.1 {angstrom} resolution.
Bibliography:USDOE
ISSN:0002-7863
1520-5126