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Nucleic Acids Research, 1985, Vol. 13, No. 24 8665-8684
© 1985

Articles

T4 RNA ligase catalyzed synthesis of base analogue-containing oligodeoxyribonucleotides and a characterization of their thermal stabilities

Catherine A. Brennan1 and Richard I. Gumport2

Department of Biochemistry, College of Medicine and School of Chemical Sciences, University of Illinois Urbana, IL 61801, USA

2To whom correspondence should be addressed.

Received November 13, 1985. Accepted November 15, 1985.

Self-complementary oligodeoxyribonucleotides containing the base analogues 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, uracil, and 5-bromouracil were synthesized by a general method that allows incorporation of the analogues at specific positions. The method uses chemically synthesized partial sequences but circumvents the need for protected base analogues by incorporating their unprotected 3',5'-bisphosphate derivatives enzymatically. T4 RNA ligase was used to add the analogues to the oligodeoxyribonucleotides with yields from 54 to greater than 95 percent. Oligodeoxyribonucleotides were joined to the oligodeoxyribonucleotides containing the analogues at their 3'-termini in yields from 22 to 81 percent. The high yields obtained in these joinings suggest that RNA ligase should be of general use for the specific incorporation of other deoxyribonucleoside analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.

1Present address: Department of Biochemistry, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642.


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