Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5'-(alpha-P-seleno)triphosphate and DNA polymerase for X-ray crystallography via MAD.
We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two alpha-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.
Carrasco
Nicolas
N
Department of Chemistry, Brooklyn College, and Program of Biochemistry and Chemistry, The Graduate School, The City University of New York, 2900 Bedford Avenue, Brooklyn, New York 11210, USA.
Huang
Zhen
Z
D018360:Crystallography, X-Ray; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005090:Exodeoxyribonucleases / Q000737:chemistry; D016566:Organoselenium Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry*; D018036:Selenium Compounds / Q000737:chemistry*; D013942:Thymine Nucleotides / Q000737:chemistry
D016566:Organoselenium Compounds; D010710:Phosphates; D018036:Selenium Compounds; D013942:Thymine Nucleotides; C484370:thymidine 5'-(alpha-P-seleno)triphosphate; C076818:selenophosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III