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

Abstract

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 deoxyribonucleotide analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.