Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers.
Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers.
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DOI：
10.1021/bi020669d
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发表时间：
2003-04
期刊：
影响因子：
2.9
通讯作者：
H. Kuhn;Yongbo Hu;M. Frank-Kamenetskii;V. Demidov
中科院分区：
文献类型：
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作者：
H. Kuhn;Yongbo Hu;M. Frank-Kamenetskii;V. Demidov
我们报告的肽核酸（PNA）指导设计的DNA切口系统，使选择性和定量切割的双链体DNA的一条链在指定的网站，从而模仿天然切口酶，并显着扩展其潜力。该系统利用嘧啶PNA通过侵入DNA双链体并暴露一条DNA链用于寡核苷酸杂交来充当特异性DNA位点的开放剂的能力。所得到的二级双链体可以作为限制酶的底物，其最终在亲本DNA中产生切口。我们证明了几种不同类型的限制性内切酶可以成功地用于我们开发的PNA辅助系统。重要的是，与常规DNA双链体上的效率相比，酶切割效率在这种人工产生的底物上基本上不受损害。我们的设计起源于大量的半合成稀有切割DNA切口酶，目前基本上不存在。此外，我们表明，双链体DNA的位点特异性PNA辅助切口可以参与滚环DNA扩增（RCA）反应。这种新的RCA格式展示了我们为DNA技术和DNA诊断提出的新型生物分子工具的实用潜力。
We report on the peptide nucleic acid (PNA)-directed design of a DNA-nicking system that enables selective and quantitative cleavage of one strand of duplex DNA at a designated site, thus mimicking natural nickases and significantly extending their potential. This system exploits the ability of pyrimidine PNAs to serve as openers for specific DNA sites by invading the DNA duplex and exposing one DNA strand for oligonucleotide hybridization. The resultant secondary duplex can act as a substrate for a restriction enzyme, which ultimately creates a nick in the parent DNA. We demonstrate that several restriction enzymes of different types could be successfully used in the PNA-assisted system we developed. Importantly, the enzyme cleavage efficiency is basically not impaired on such artificially generated substrates, compared with the efficiency on regular DNA duplexes. Our design originates a vast class of semisynthetic rare-cleaving DNA nickases, which are essentially absent at present. In addition, we show that the site-specific PNA-assisted nicking of duplex DNA can be engaged in a rolling-circle DNA amplification (RCA) reaction. This new RCA format demonstrates the practical potential of the novel biomolecular tool we propose for DNA technology and DNA diagnostics.