Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site.
The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.
D002851:Chromatography, High Pressure Liquid; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D063086:Organothiophosphates; D013237:Stereoisomerism; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D063086:Organothiophosphates; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI