Amplification of 4'-thioDNA in the presence of 4'-thio-dTTP and 4'-thio-dCTP, and 4'-thioDNA-directed transcription in vitro and in mammalian cells
Amplification of 4'-thioDNA in the presence of 4'-thio-dTTP and 4'-thio-dCTP, and 4'-thioDNA-directed transcription in vitro and in mammalian cells
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DOI：
10.1021/ja075953c
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发表时间：
2007-12-19
影响因子：
15
通讯作者：
Matsuda, Akira
中科院分区：
文献类型：
--
作者：
Inoue, Naonori;Shionoya, Aki;Matsuda, Akira
本文报道了4‘-硫代-dTTP(1)和4’-硫代-dCTP(2)的合成及其对聚合酶链式反应的敏感性。KOD-DASH DNA聚合酶在适宜的条件下能扩增出双链4‘-硫代DNA。通过该聚合酶链式反应的研究表明，在1和/或2的存在下，不仅DNA引导的4‘-硫代DNA合成，而且4’-硫代DNA的4‘-硫代DNA的合成也发生了。在本实验中，4‘-硫代DNA作为模板提供了与天然DNA模板相比产率为38-49%的RNA。我们进一步研究了哺乳动物细胞中是否存在转录。因此，不仅天然DNA被转录，而且4‘-硫代DNA也被转录成通过RNAi机制显示基因沉默效应的shRNA。
Herein we describe the synthesis of 4'-thio-dTTP (1) and 4'-thio-dCTP (2) and their susceptibility for PCR amplification. Double stranded 4'-thioDNAs were amplified sufficiently by KOD dash DNA polymerase under appropriate conditions. Through this PCR study, it was shown that not only DNA-directed 4'-thioDNA synthesis but also 4'-thioDNA-directed 4'-thioDNA synthesis occurred in the presence of 1 and/or 2. We then tested the ability of the resulting 4'-thioDNAs to act as templates for transcription. In this experiment, the 4'-thioDNAs as templates afforded RNA in 38-49% yield relative to the case of the natural DNA template. We further investigated whether transcription occurred in mammalian cells. As a result, not only natural DNA but also 4'-thioDNAs were transcribed to afford a shRNA which showed gene-silencing effects via RNAi machinery.