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Nucleic Acids Research, 1987, Vol. 15, No. 18 7451-7462
© 1987

Articles

Synthesis and properties of oligodeoxynucleotides with an AP site at a preselected position

Gregory R. Stuart and Robert W. Chambers

Department of Biochemistry, Dalhousie University Halifax, Nova Scotia B3H 4H7, Canada

Received July 31, 1987. Revised August 24, 1987. Accepted August 24, 1987.

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(TTTTTTT), where r = deoxyribose, and d(AAAAGTTprpAAAACAT). KM and Vmax are: d(TTTUTTTT), 7.3 x 10–9 M and 2.0 x 10–9 µmol/min; d(AAAAGTTUAAAACAT), 1.5 x 10–8 M and 6.4 x 10–9 µmol / min. Both d(AAAAGTTprpAAAACAT) and d(TTTprpTTTT) undergo rapid ß-elimination in 1 M piperidine at 25° 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'{uparrow}5' exonuclease activity of snake venom phosphodiesterase. Endonucleolytic hydrolysis of the penultimate phosphodiestcr occurs removing pTpr1 and generating a normal 3'-OH end. In 1 M piperidine at 90° two ß-eliminations occur producing the oligonucleotides d(AAAAGTTp) and d(pAAAACAT) from d(AAAAGTTprpAAAACAT); d(TTTp) and d(pTTTT) from d(TTTprpTTTT).


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