Kuhn H, Demidov V V, Nielsen P E, Frank-Kamenetskii M D
Center for Advanced Biotechnology, Department of Biomedical Engineering, Boston University, 36 Cummington St, Boston, MA, 02215, USA.
J Mol Biol. 1999 Mar 12;286(5):1337-45. doi: 10.1006/jmbi.1998.2578.
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 are 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. In 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.
我们研究了肽核酸夹（双肽核酸）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肽核酸与dsDNA上匹配和错配靶标的初始结合速率之比。双肽核酸由两个通过柔性接头连接的同嘧啶肽核酸寡聚物组成。已知它们在与dsDNA复合时会形成P环，P环由一个[肽核酸]2-DNA三链体和被置换的DNA链组成。我们在此报告，对于仅由C和T碱基组成的双肽核酸，在中性pH范围内，其结合速率和动力学特异性对pH具有很强的依赖性。在pH 6.9时，结合特异性达到非常尖锐且高的最大值。相比之下，如果双肽核酸中两个肽核酸寡聚物之一中的所有胞嘧啶碱基都被假异胞嘧啶碱基（J碱基）取代，而J碱基形成三链体不需要质子化，则观察到P环形成速率和特异性对pH的依赖性较弱。对数据的理论分析表明，对于含（C + T）的双肽核酸，肽核酸与dsDNA结合的第一步中间步骤是通过双链靶标与双肽核酸的一个寡聚物之间的Hoogsteen配对发生的。之后，链入侵通过第二个双肽核酸寡聚物与靶标DNA的同嘌呤链之间的沃森-克里克配对发生，从而最终形成P环。含（C/J + T）的双肽核酸的数据表明，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（C/J + T）的肽核酸结构在体内使用可能具有优势。
