Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids
Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids
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DOI：
10.1073/pnas.092127999
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发表时间：
2002-04-30
影响因子：
11.1
通讯作者：
Frank-Kamenetskii, MD
中科院分区：
文献类型：
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作者：
Demidov, VV;Protozanova, E;Frank-Kamenetskii, MD
如果两个相互互补的混合碱基肽核酸 (PNA) 寡聚体中的腺嘌呤和胸腺嘧啶分别被二氨基嘌呤和硫尿嘧啶取代，则产生所谓的假互补 PNA (pcPNA)。最近，pcPNA 对已证明能够通过双链体侵入（螺旋侵入）形成非常稳定的 PNA-DNA 链置换复合物，从而高度选择性地靶向双链 DNA (dsDNA) 上的几乎任何指定位点。 pcPNA 的这些特性使其成为独特且非常有前途的配体，能够阻止 DNA 结合蛋白接近 dsDNA。为了阐明 pcPNA 识别序列特异性 dsDNA 的非序列限制机制，我们通过凝胶迁移实验研究了在不同温度下相应 PNA-DNA 复合物形成的动力学。同时，通过混合曲线（作业图）和热熔解实验测定了 pcPNA 寡聚物可能的自杂交条件。数据表明，在生理温度（大约 37 摄氏度）下，平衡向相应 pcPNA 彼此配对的方向移动。这一发现解释了 37°C 下 pcPNA 侵入 dsDNA 的速率在亚微摩尔范围内的线性浓度依赖性。在升高的温度（> 50°C）下，相当不稳定的 pcPNA 双链体解离，产生预期的 pcPNA 入侵速率对 PNA 浓度的二次依赖性。 pcPNA 对的聚阳离子特征，携带双倍数量的质子化末端 PNA 残基，通常用于增加 PNA 溶解度和结合亲和力，也解释了在较高 PNA 浓度下观察到的 pcPNA 入侵的自我抑制。 pcPNA双链体发生熔化，积分转变焓范围为-235至-280 kJ.mol(-1)，导致携带四种不同核碱基的pcPNA螺旋侵入时发现异常高的活化能，约为150 kJ.mol(-1)。提出了 pcPNA 螺旋入侵的简化动力学模型，该模型解释了 pcPNA 与 dsDNA 结合的所有异常特征。我们的研究结果对于合理使用 pcPNA 具有重要意义。
If adenines and thymines in two mutually complementary mixed-base peptide nucleic acid (PNA) oligomers are substituted with diaminopurines and thiouracils, respectively, so-called pseudo-complementary PNAs (pcPNAs) are created. Pairs of pcPNAs have recently demonstrated an ability to highly selectively target essentially any designated site on double-stranded DNA (dsDNA) by forming very stable PNA-DNA strand-displacement complexes via double duplex invasion (helix invasion). These properties of pcPNAs make them unique and very promising ligands capable of denying the access of DNA-binding proteins to dsDNA. To elucidate the sequence-unrestricted mechanism of sequence-specific dsDNA recognition by pcPNAs, we have studied the kinetics of formation of corresponding PNA-DNA complexes at various temperatures by the gel-shift assay. In parallel, the conditions for possible self-hybridization of pcPNA oligomers have been assayed by mixing curve (Job plot) and thermal melting experiments. The data indicate that, at physiological temperatures (approximate to37degreesC), the equilibrium is shifted toward the pairing of corresponding pcPNAs with each other. This finding explains a linear concentration dependence, within the submicromolar range, of the pcPNA invasion rate into dsDNA at 37degreesC. At elevated temperatures (>50degreesC), the rather unstable pcPNA duplexes dissociate, yielding the expected quadratic dependence for the rate of pcPNA invasion on the PNA concentration. The polycationic character of pcPNA pairs, carrying the duplicated number of protonated terminal PNA residues commonly used to increase the PNA solubility and binding affinity, also explains the self-inhibition of pcPNA invasion observed at higher PNA concentrations. Melting of pcPNA duplexes occurs with the integral transition enthalpies ranged from -235 to -280 kJ.mol(-1), contributing to an anomalously high activation energy of approximate to150 kJ.mol(-1) found for the helix invasion of pcPNAs carrying four different nucleobases. A simplified kinetic model for pcPNAs helix invasion is proposed that interprets all unusual features of pcPNAs binding to dsDNA. Our findings have important implications for rational use of pcPNAs.