Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA
Kinetic sequence discrimination of cationic bis-PNAs upon targeting of double-stranded DNA
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DOI：
10.1093/nar/26.2.582
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发表时间：
1998-01-15
影响因子：
14.9
通讯作者：
Nielsen, PE
中科院分区：
文献类型：
--
作者：
Kuhn, H;Demidov, VV;Nielsen, PE
报道了阳离子假异胞嘧啶连接的同嘧啶肽核酸（bis-PNA）与双链DNA（dsDNA）中完全匹配和单错配的十嘌呤靶的链置换结合动力学。通过凝胶迁移率变动测定监测PNA-dsDNA复合物的形成，并且在所有研究情况下均遵守结合的伪一级动力学。PNA与dsDNA结合的动力学特异性（定义为与匹配和错配靶标结合的初始速率的比率）随着离子强度的增加而增加，而双-PNA-dsDNA复合物形成的表观速率常数呈指数下降。在非常低的离子强度下，两个等电荷的双-具有相同的核碱基序列但不同的接头并因此具有不同的三个正电荷位置的PNA在它们的结合特异性上相差一个数量级，在适当的实验条件下，双PNA靶向dsDNA的动力学特异性高达300。因此，在Hoogsteen链中含有假异胞嘧啶（J碱基）的多电荷阳离子双PNA结合增强的结合亲和力也表现出非常高的序列特异性，从而使得此类试剂对于双链体DNA的序列特异性靶向极其有效。
Strand displacement binding kinetics of cationic pseudoisocytosine-containing linked homopyrimidine peptide nucleic acids (bis-PNAs) to fully matched and singly mismatched decapurine targets in double-stranded DNA (dsDNA) are reported. PNA-dsDNA complex formation was monitored by gel mobility shift assay and pseudo-first order kinetics of binding was obeyed in all cases studied. The kinetic specificity of PNA binding to dsDNA, defined as the ratio of the initial rates of binding to matched and mismatched targets, increases with increasing ionic strength, whereas the apparent rate constant for bis-PNA-dsDNA complex formation decreases exponentially Surprisingly, at very low ionic strength two equally charged bis-PNAs which have the same sequence of nucleobases but different linkers and consequently different locations of three positive charges differ in their specificity of binding by one order of magnitude, Under appropriate experimental conditions the kinetic specificity for bis-PNA targeting of dsDNA is as high as 300, Thus multiply charged cationic bis-PNAs containing pseudoisocytosines (J bases) in the Hoogsteen strand combined with enhanced binding affinity also exhibit very high sequence specificity, thereby making such reagents extremely efficient for sequence-specific targeting of duplex DNA.