The preparative synthesis of oligodeoxyribonucleotides using RNA ligase

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Abstract
The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reactions using from 18 to 200 nmol of substrates in which a single 2′-deoxyribonucleoside 3′,5′-bisphosphate was added to an oligodeoxyribonucleotide resulted in yields from 13 to 95%. When two oligodeoxyribonucleotides were similarly joined using RNA ligase, the yields ranged from 10 to 50%. Although the reactions contained high concentrations of enzyme and were incubated from 5 to 21 days, there was little degradation of either substrates or products. We have also characterized an unusual product which arises when 3′-phosphate terminated oligodeoxyribonucleotides are incubated with RNA ligase and high concentrations of ATP. This product has an adenylyl group linked to the 3′-phosphate by an anhydride bond. The mechanistic and synthetic implications of forming this product are discussed.