Origin of high fidelity in target-sequence recognition by PNA-Ce(IV)/EDTA combinations as site-selective DNA cutters.
Origin of high fidelity in target-sequence recognition by PNA-Ce(IV)/EDTA combinations as site-selective DNA cutters.
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DOI：
10.1021/ja808290e
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发表时间：
2009-02
影响因子：
15
通讯作者：
Y. Miyajima;Takumi Ishizuka;Yoji Yamamoto;J. Sumaoka;M. Komiyama
中科院分区：
文献类型：
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作者：
Y. Miyajima;Takumi Ishizuka;Yoji Yamamoto;J. Sumaoka;M. Komiyama
伪互补肽核酸（pcPNA）的双链体入侵是识别双链DNA中特定位点的最重要策略之一（Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11804-11808）。这种策略最近被用来开发人工限制性 DNA 切割器 (ARCUT)，用于双链 DNA 的位点选择性切割，其中 PNA 双链体入侵形成的热点被 Ce(IV)/EDTA 水解 (Nat. Protoc. 2008, 3, 655-662)。本文展示了 PNA-Ce(IV)/EDTA 组合如何以及在何处识别双链 DNA 中的靶序列以进行位点选择性断裂。评估入侵过程和整个分裂过程中的错配识别活动。当两种 pcPNA 添加剂与 DNA 的每条链完全互补时，正如预期的那样，位点选择性断裂是最有效的。当入侵位点的一个 DNA 碱基对与另一碱基对交换时（这会在 pcPNA 和 DNA 之间引入错配），ARCUT 的位点选择性断裂显着减少。中央双侵入区域（或附近）的错配尤其致命，表明该区域的 DNA 碱基与 pcPNA 链的 Watson-Crick 配对对于精确识别目标序列至关重要。双链体侵袭过程的凝胶迁移测定和解链温度测量均已证实该过程的保真度主要控制 DNA 断裂的保真度。根据这些系统分析，涉及两个 15 聚体 pcPNA 的典型 ARCUT 可以精确识别底物 DNA 中的 14-16 个碱基对。这种非凡的保真度是在与细胞中的值相似的相当高的盐浓度下实现的。
Double-duplex invasion of pseudocomplementary peptide nucleic acid (pcPNA) is one of the most important strategies for recognizing a specific site in double-stranded DNA (Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11804-11808). This strategy has recently been used to develop artificial restriction DNA cutters (ARCUTs) for site-selective scission of double-stranded DNA, in which a hot spot formed by double-duplex invasion of PNA was hydrolyzed by Ce(IV)/EDTA (Nat. Protoc. 2008, 3, 655-662). The present paper shows how and where the target sequence in double-stranded DNA is recognized by the PNA-Ce(IV)/EDTA combinations for site-selective scission. The mismatch-recognizing activities in both the invasion process and the whole scission process are evaluated. When both pcPNA additives are completely complementary to each strand of the DNA, site-selective scission is the most efficient, as expected. Upon exchange of one DNA base pair at the invasion site with another base pair, which introduces mismatches between the pcPNAs and the DNA, the site-selective scission by the ARCUT is notably diminished. Mismatches in (or near) the central double-invasion region are especially fatal, showing that Watson-Crick pairings of the DNA bases in this region with the pcPNA strands are essential for precise recognition of the target sequence. Both gel-shift assays and melting temperature measurements on the double-duplex invasion process have confirmed that the fidelity in this process primarily governs the fidelity of the DNA scission. According to these systematic analyses, the typical ARCUT involving two 15-mer pcPNAs precisely recognizes 14-16 base pairs in substrate DNA. This remarkable fidelity is accomplished at rather high salt concentrations that are similar to the values in cells.