Hinton D M, Brennan C A, Gumport R I
Nucleic Acids Res. 1982 Mar 25;10(6):1877-94. doi: 10.1093/nar/10.6.1877.
The synthesis of nmol quantities of defined sequences of oligodeoxyribonucleotides using T4 RNA ligase has been demonstrated. Reacting 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.
已证明使用T4 RNA连接酶可合成纳摩尔量的特定寡脱氧核糖核苷酸序列。以18至200纳摩尔的底物进行反应，其中将单个2'-脱氧核糖核苷3',5'-双磷酸添加到寡脱氧核糖核苷酸中，产率为13%至95%。当使用RNA连接酶以类似方式连接两个寡脱氧核糖核苷酸时，产率范围为10%至50%。尽管反应中含有高浓度的酶，且孵育5至21天，但底物或产物几乎没有降解。我们还鉴定了一种不寻常的产物，当3'-磷酸末端的寡脱氧核糖核苷酸与RNA连接酶和高浓度的ATP一起孵育时会产生这种产物。该产物具有通过酸酐键与3'-磷酸相连的腺苷酰基。讨论了形成该产物的机制和合成意义。
