Ashley G W, Kushlan D M
Department of Chemistry, Northwestern University, Evanston, Illinois 60208.
Biochemistry. 1991 Mar 19;30(11):2927-33. doi: 10.1021/bi00225a028.
The chemical synthesis of DNA dumbbells is investigated by using two sequences, cyclo-d(GCG-T4-CGCCGC-T4-GCG) and cyclo-d(TTCC-T4-GGAATTCC-T4-GGAA). This method readily and inexpensively yields multimicromole quantities of circular DNA, allowing detailed structural and physical studies to be carried out. Linear oligomers of sequence d(GCG-T4-CGCCGC-T4-GCG) having either 3'- or 5'-phosphates were cyclized in 40% and 67% isolated yield, respectively, by using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide. Formation of the circular product is confirmed by a 28 degrees C increase in the optical melting temperature, anomalously rapid electrophoretic migration, sequential nuclear Overhauser enhancements between protons of G1 and G20, and observed nuclear coupling between the ligated phosphorus and protons of both G1 and G20. cyclo-d(TTCC-T4-GGAATTCC-T4-GGCC) was synthesized from the corresponding linear 3'-phosphate in 80% yield by the same procedure. Chemical ligation is most effective for 3'-phosphorylated nick sites flanked by two purine bases.
通过使用两个序列，即环化-d（GCG-T4-CGCCGC-T4-GCG）和环化-d（TTCC-T4-GGAATTCC-T4-GGAA），对DNA哑铃结构的化学合成进行了研究。该方法能够轻松且廉价地产生多微摩尔量的环状DNA，从而可以进行详细的结构和物理研究。分别使用1-[3-(二甲氨基)丙基]-3-乙基碳二亚胺，使具有3'-或5'-磷酸基团的序列d（GCG-T4-CGCCGC-T4-GCG）的线性寡聚物环化，分离产率分别为40%和67%。通过光学熔解温度升高28℃、异常快速的电泳迁移、G1和G20质子之间的连续核Overhauser增强以及观察到连接的磷与G1和G20质子之间的核耦合，证实了环状产物的形成。通过相同的程序，由相应的线性3'-磷酸合成环化-d（TTCC-T4-GGAATTCC-T4-GGCC），产率为80%。化学连接对于两侧有两个嘌呤碱基的3'-磷酸化切口位点最为有效。
