Combined triplex/duplex invasion of double-stranded DNA by "tail-clamp" peptide nucleic acid
Combined triplex/duplex invasion of double-stranded DNA by "tail-clamp" peptide nucleic acid
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DOI：
10.1021/bi0351918
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发表时间：
2003-12-02
期刊：
影响因子：
2.9
通讯作者：
Nielsen, PE
中科院分区：
文献类型：
--
作者：
Bentin, T;Larsen, HJ;Nielsen, PE
设计了由一个短的（六聚体）同嘧啶三链体形成结构域和一个（十聚体）混合序列双链体形成延伸部分组成的“尾夹”肽核酸（PNAs）。通过熔解温度（T - m）测量确定，与缺乏双链体形成延伸部分的肽核酸相比，尾夹肽核酸与单链DNA的结合显著增强。通过高锰酸盐探测分析，其与双链（ds）DNA的结合是通过三链体和双链体联合侵入发生的。此外，半数抑制浓度（C - 50）测量显示尾夹肽核酸始终能更有效地结合双链DNA靶标，动力学实验表明这是由于此类复合物的解离速率大幅降低。增加肽核酸的净电荷也提高了结合效率，但出乎意料的是，无尾夹肽核酸的这种增加比尾夹肽核酸更为显著。最后，将尾夹肽核酸的三链体侵入部分缩短至五个残基是可行的，但四个碱基不足以产生可检测的双链DNA结合。这些结果验证了尾夹肽核酸的概念，并扩展了P - 环技术的应用。
"Tail-clamp" PNAs composed of a short (hexamer) homopyrimidine triplex forming domain and a (decamer) mixed sequence duplex forming extension have been designed. Tail-clamp PNAs display significantly increased binding to single-stranded DNA compared with PNAs lacking a duplex-forming extension as determined by T-m measurements. Binding to double-stranded (ds) DNA occurred by combined triplex and duplex invasion as analyzed by permanganate probing. Furthermore, C-50 measurements revealed that tail-clamp PNAs consistently bound the dsDNA target more efficiently, and kinetics experiments revealed that this was due to a dramatically reduced dissociation rate of such complexes. Increasing the PNA net charge also increased binding efficiency, but unexpectedly, this increase was much more pronounced for tailless-clamp PNAs than for tail-clamp PNAs. Finally, shortening the tail-clamp PNA triplex invasion moiety to five residues was feasible, but four bases were not sufficient to yield detectable dsDNA binding. The results validate the tail-clamp PNA concept and expand the applications of the P-loop technology.