Nature Methods
- 3, 729 - 735 (2006)
Published online: 23 August 2006; | doi:10.1038/nmeth915
An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNAIchiro Hirao1, 2, Michiko Kimoto1, Tsuneo Mitsui1, 2, Tsuyoshi Fujiwara1, 5, Rie Kawai1, Akira Sato1, Yoko Harada1 & Shigeyuki Yokoyama1, 3, 41
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230–0045, Japan
2
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
3
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
4
RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
5
Present address: Nanotechnology Research Center, Fujitsu Laboratories, 10-1 Morinosato-Wakamiya, Atsugi, Kanagawa 243-0197, Japan.
Correspondence should be addressed to Ichiro Hirao ihirao@riken.jp or Shigeyuki Yokoyama yokoyama@biochem.s.u-tokyo.ac.jp 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,  -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.
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