An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA
An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA
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DOI：
10.1038/nmeth915
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发表时间：
2006-09-01
期刊：
影响因子：
48
通讯作者：
Yokoyama, Shigeyuki
中科院分区：
文献类型：
--
作者：
Hirao, Ichiro;Kimoto, Michiko;Yokoyama, Shigeyuki
在核酸中加入特定部位的额外成分的方法可以成为创造具有更多功能的DNA和RNA分子的强大工具。我们提出了一种非自然碱基对系统，在该系统中，含有非自然碱基对的DNA可以被扩增，并作为模板，通过转录将碱基类似物结合到RNA中。非天然碱基对是通过碱基之间特定的疏水形状互补形成的，但缺乏氢键相互作用。在复制中，这一非天然碱基对与常见的三磷酸盐和修饰的三磷酸盐，即伽马-氨基三磷酸盐，作为3‘到5’核酸外切酶熟练的DNA聚合酶的底物，具有很高的选择性，允许进行PCR扩增。在转录过程中，非自然的碱基对互补介导了这些碱基底物及其类似物，如生物素化底物，通过T7RNA聚合酶(RNAP)掺入RNA。有了这个系统，功能成分可以被定位地整合到一个大的RNA分子中。
Methods for the site-specific incorporation of extra components into nucleic acids can be powerful tools for creating DNA and RNA molecules with increased functionality. We present an unnatural base pair system in which DNA containing an unnatural base pair can be amplified and function as a template for the site-specific incorporation of base analog substrates into RNA via transcription. The unnatural base pair is formed by specific hydrophobic shape complementation between the bases, but lacks hydrogen bonding interactions. In replication, this unnatural base pair exhibits high selectivity in combination with the usual triphosphates and modified triphosphates, gamma-amidotriphosphates, as substrates of 3' to 5' exonuclease-proficient DNA polymerases, allowing PCR amplification. In transcription, the unnatural base pair complementarity mediates the incorporation of these base substrates and their analogs, such as a biotinylated substrate, into RNA by T7 RNA polymerase (RNAP). With this system, functional components can be site-specifically incorporated into a large RNA molecule.