Enzymatic Synthesis of Phosphoroselenoate DNA Using Thymidine 5‘-(α-P- seleno)triphosphate and DNA Polymerase for X-ray Crystallography via MAD

• Published in: Journal of the American Chemical Society Vol. 126; no. 2; pp. 448 - 449
• Main Authors: Carrasco, Nicolas, Huang, Zhen
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 21.01.2004
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Crystallography, X-Ray; DNA - chemical synthesis; DNA - chemistry; Dna, deoxyribonucleoproteins; DNA-Directed DNA Polymerase - chemistry; Exodeoxyribonucleases - chemistry; Fundamental and applied biological sciences. Psychology; Nucleic acids; Organoselenium Compounds - chemical synthesis; Organoselenium Compounds - chemistry; Phosphates - chemistry; Selenium Compounds - chemistry; Thymine Nucleotides - chemistry; Enzymatic synthesis; Nucleotidyltransferases; Molecular structure; Enzyme; Transferases; Nucleic acid synthesis; X ray diffraction; Pyrimidine nucleoside; DNA-directed DNA polymerase; Diastereoselectivity; Crystalline structure; Organic selenophosphate
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0383221

We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two α-Se-TTP diastereomers (Sp and Rp) and DNA polymerase. The experimental results indicate that Klenow equally recognizes the two individual diastereomers at the same level as natural TTP. The incorporations of the PSe groups at the expected sites have been confirmed by the digestion resistance to exonuclease III, and the different patterns of the digestion resistance of DNA I and II indicate the configurational differences of the PSe centers (Sp or Rp). Unlike chemical synthesis, which is limited to short DNAs and where the separation of the PSe DNA diastereomers is necessary, this enzymatic method can be used to prepare longer DNAs without diastereomer separation. This quantitative enzymatic approach is particular valuable for the synthesis of longer DNAs with multiple PSe groups in large scale for their X-ray crystal structure determination by the MAD phasing technique.