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首页> 外文期刊>Nucleic Acids Research >Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates
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Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates

机译：吡咯-咪唑-环丙吡咯并吲哚偶联物对质粒DNA的限制性核酸内切酶水解的序列特异性保护

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摘要

The pyrrole-imidazole (Py-Im) triamide-cyclopropa-pyrroloindole (CP) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT 1 (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHll (5'-G-CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHll. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco521 (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQB1 PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by Dral(3'-TTT_AAA-3') was not inhibited by 5 μM conjugate 14. That ImIPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBl PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as wasrequired to protect supercoiled DNA from hydrolysis.
机译：合成了吡咯-咪唑（Py-Im）三酰胺-环丙烷-吡咯并吲哚（CP）共轭物ImPyImLDu86（7）和ImImPyLDu86（14），并研究了其烷基化活性和限制性内切酶对DNA水解的抑制作用。测序凝胶分析表明，缀合物7和14分别在5'-CGCGCG-3'和5'-PyGGCCPu-3'处特异性烷基化了DNA。琼脂糖凝胶电泳表明，超螺旋质粒pSPORT 1（4109 bp）与缀合物7的孵育有效地抑制了BssHll的水解（5'-G-CGCGC-3'），而缀合物14对此水解没有影响。这些结果表明缀合物7序列特异性地抑制了BssH11对DNA的水解。通过抑制结合物14处理的pQB1 PGK（5387 bp）的Eco521（5'-C_GGCCG-3'）水解，进一步证实了Py-Im三酰胺-CPI结合物的序列特异性烷基化。与之形成鲜明对比的是，5μM偶联物14不会抑制Dral（3'-TTT_AAA-3'）对pQB1 PGK的水解。ImIPy不会抑制pQB1 PGK的水解，表明共价键形成对于抑制是必要的。使用线性pQB1 PGK进行的类似实验获得了对DNA的保护程度，其结合物14的浓度约为保护超螺旋DNA免于水解的一半。

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