Hirao Ichiro, Kimoto Michiko, Mitsui Tsuneo, Fujiwara Tsuyoshi, Kawai Rie, Sato Akira, Harada Yoko, Yokoyama Shigeyuki
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Nat Methods. 2006 Sep;3(9):729-35. doi: 10.1038/nmeth915.
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.
将额外组分位点特异性地掺入核酸的方法可能是用于创建具有增强功能的DNA和RNA分子的强大工具。我们提出了一种非天然碱基对系统，其中含有非天然碱基对的DNA可以被扩增，并作为模板通过转录将碱基类似物底物位点特异性地掺入RNA中。该非天然碱基对由碱基之间特定的疏水形状互补形成，但缺乏氢键相互作用。在复制过程中，这种非天然碱基对与通常的三磷酸酯和修饰的三磷酸酯（γ-氨基三磷酸酯）结合时，作为具有3'至5'外切核酸酶活性的DNA聚合酶的底物，表现出高选择性，从而允许进行PCR扩增。在转录过程中，非天然碱基对互补性介导这些碱基底物及其类似物（如生物素化底物）被T7 RNA聚合酶（RNAP）掺入RNA中。利用该系统，功能组分可以位点特异性地掺入到一个大的RNA分子中。
