An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA
An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA
复制标题
DOI：
10.1006/jmbi.1998.2578
复制
发表时间：
1999-03-12
影响因子：
5.6
通讯作者：
Frank-Kamenetskii, MD
中科院分区：
文献类型：
--
作者：
Kuhn, H;Demidov, VV;Frank-Kamenetskii, MD
我们研究了肽核酸钳（bis-PNAs）与双链DNA（dsDNA）结合的机制和动力学特异性。动力学特异性定义为PNA与dsDNA上匹配和错配靶标结合的初始速率的比率。Bis-PNA由通过柔性接头连接的两个同型嘧啶PNA寡聚体组成。当与dsDNA复合时，它们形成P环，其由[PNA]（2）-DNA三链体和置换的DNA链组成。我们在这里报告一个非常强的pH值依赖性，在中性pH范围内，结合率和动力学特异性的双PNA组成的只有C和T基地。结合的特异性在pH 6.9时达到非常尖锐和高的最大值。相反，如果双PNA内的两个PNA寡聚体之一中的所有胞嘧啶碱基被假异胞嘧啶碱基（J碱基）取代，其不需要质子化以形成三链体，则P环形成的速率和特异性对pH的弱依赖性被消除。PNA与dsDNA结合的中间步骤通过双链体靶标和双PNA的一个寡聚体之间的Hoogsteen配对发生。之后，通过第二双PNA寡聚体和靶DNA的高嘌呤链之间的沃森-克里克配对发生链侵入，从而导致最终形成P-环。含有（C/J + T）的双PNA的数据表明，其在中性pH下对dsDNA的高亲和力不会严重损害结合的动力学特异性。这些发现支持了早期的预期，即含有（C/J + T）的PNA结构可能有利于体内使用。(C)北京：科学出版社.
We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they ape known to form P-loops, which consist of a [PNA](2)-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding; rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. Ln contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed.A theoretical analysis of the data suggests that for (C + T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J + T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J + T)-containing PNA constructions may be advantageous for use in vivo. (C) 1999 Academic Press.