KINETICS AND MECHANISM OF POLYAMIDE (PEPTIDE) NUCLEIC-ACID BINDING TO DUPLEX DNA
KINETICS AND MECHANISM OF POLYAMIDE (PEPTIDE) NUCLEIC-ACID BINDING TO DUPLEX DNA
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DOI：
10.1073/pnas.92.7.2637
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发表时间：
1995-03-28
影响因子：
11.1
通讯作者：
NIELSEN, PE
中科院分区：
文献类型：
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作者：
DEMIDOV, VV;YAVNILOVICH, MV;NIELSEN, PE
为了阐明同型嘧啶聚酰胺（“肽”）核酸（PNA）识别双链DNA （dsDNA）导致链位移的机制，研究了序列特异性PNA/DNA结合的动力学，通过凝胶阻滞和核酸酶S1裂解实验监测了这种结合随时间的变化，实验动力学曲线符合准一级动力学，并且准一级速率常数k（ps）与PNA浓度P、服从幂定律k(ps)，类似于P-gamma， 2 < gamma < 3。十聚体PNA与双链DNA靶位点结合的k（ps）值比正确位点慢数百倍。提出了PNA/DNA结合的详细动力学方案，包括链入侵反应的两个主要步骤：(i) PNA在dsDNA上的结合位点瞬间部分打开，一个PNA分子结合形成中间的PNA/DNA双链，（ii）形成非常稳定的PNA(2)/DNA三链。对所提出的动力学方案进行了简单的理论处理，在所提出的动力学方案框架下解释我们的实验数据得出以下结论：识别的序列特异性基本上是在该过程的“搜索”步骤中提供的，该步骤包括一个PNA分子与双链DNA互补链之间高度可逆的瞬态双链形成，而另一个DNA链被移位。这个搜索步骤之后是通过PNA(2)/DNA三倍体形成的几乎不可逆的“锁定”步骤。提出的机制解释了同型嘧啶PNA与dsDNA的结合如何满足两个明显相互矛盾的特征：高序列特异性的结合和正确和错配的PNA/DNA复合物的显著稳定性。
To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide (''peptide'') nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied, The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays, The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first order rate constant, k(ps), on PNA concentration, P, obeys a power law k(ps) similar to P-gamma with 2 < gamma < 3. The k(ps) values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site, A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA(2)/DNA tripler, A simple theoretical treatment of the proposed kinetic scheme is performed, The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions, The se quence specificity of the recognition is essentially provided at the ''search'' step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced, This search step is followed by virtually irreversible ''locking'' step via PNA(2)/DNA tripler formation, The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes,