Synthesis and properties of oligodeoxynucleotides with an AP site at a preselected position.
A general synthesis of a deoxyoligonucleotide with an AP site at a preselected sequence is described. Deoxyuridine is introduced during routine oligonucleotide syntheses of d(TTTUTTTT) and d(AAAAGTTUAAAACAT). Treatment with uracil DNA-glycosylase produces d(TTTrTTTT), where r = deoxyribose, and d(AAAAGTTprpAAAACAT). KM and Vmax are: d(TTTUTTTT), 7.3 X 10(-9)M and 2.0 X 10(-9) mumol/min; d(AAAAGTTUAAAACAT), 1.5 X 10(-8) M and 6.4 X 10(-9) mumol/min. Both d(AAAAGTTprpAAAACAT) and d(TTTprpTTTT) undergo rapid beta-elimination in 1 M piperidine at 25 degrees giving two oligonucleotide fragments, d(AAAAGTTpr') and d(pAAAACAT), where r' = -O-CH2-CHOH-CH=CH-CHO (or its hemiacetal form). The fragment, d(AAAAGTTpr'), which can be isolated by reverse phase chromatography, is resistant to the 3'----5' exonuclease activity of snake venom phosphodiesterase. Endonucleolytic hydrolysis of the penultimate phosphodiester occurs removing pTpr' and generating a normal 3'-OH end. In 1 M piperidine at 90 degrees two beta-eliminations occur producing the oligonucleotides d(AAAAGTTp) and d(pAAAACAT) from d(AAAAGTTprpAAAACAT); d(TTTp) and d(pTTTT) from d(TTTprpTTTT).
Stuart
G R
GR
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Chambers
R W
RW
D001483:Base Sequence; D004249:DNA Damage; D045647:DNA Glycosylases; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D014498:Uracil / Q000378:metabolism; D051981:Uracil-DNA Glycosidase
D009838:Oligodeoxyribonucleotides; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase