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An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA

Nature Methods volume 3, pages 729–735 (2006)Cite this article

Abstract

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, γ-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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Figure 1: An unnatural base pair system for specific replication and transcription.
Figure 2: Single-nucleotide insertion and primer extension with the Ds-Pa base pair system.
Figure 3: DNA sequencing and PCR amplification of the DNA fragments containing the Ds-Pa pair.
Figure 4: Site-specific biotinylation of RNA molecules by T7 transcription using the Ds-Pa pair.

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Acknowledgements

This work was funded by the RIKEN Structural Genomics/Proteomics Initiative, the National Project on Protein Structural and Functional Analyses, Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Grant-in-Aid for Scientific Research (KAKENHI 15350097), and by the Program of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO).

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Author notes

  1. Tsuyoshi Fujiwara

    Present address: Nanotechnology Research Center, Fujitsu Laboratories, 10-1 Morinosato-Wakamiya, Atsugi, Kanagawa, 243-0197, Japan

Authors and Affiliations

  1. Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230–0045, Kanagawa, Japan

    Ichiro Hirao, Michiko Kimoto, Tsuneo Mitsui, Tsuyoshi Fujiwara, Rie Kawai, Akira Sato, Yoko Harada & Shigeyuki Yokoyama

  2. Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan

    Ichiro Hirao & Tsuneo Mitsui

  3. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Shigeyuki Yokoyama

  4. RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo, 679-5148, Japan

    Shigeyuki Yokoyama

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  1. Ichiro Hirao
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Contributions

I.H. conceived and designed the study, supervised the work and prepared samples; M.K. contributed to the biological idea and performed experiments; T.M., T.F. and A.S. performed chemical synthesis; R.K. and Y.H. performed biological experiments; S.Y. conceived the study and supervised the work.

Note: Supplementary information is available on the Nature Methods website.

Corresponding authors

Correspondence to Ichiro Hirao or Shigeyuki Yokoyama.

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Competing interests

I.H. and S.Y. have filed a patent in Japan (2005-356883) covering some of the information described in this article.

Supplementary information

Supplementary Fig. 1

Dye terminator sequencing of the PCR production from DNA1. (PDF 689 kb)

Supplementary Fig. 2

Dye terminator sequencing of the PCR production from DNA fragments. (PDF 2520 kb)

Supplementary Fig. 3

T7 transcription mediated by the Ds-Pa pairing. (PDF 98 kb)

Supplementary Table 1

Steady-state kinetic parameters for insertion of single nucleotides into a template-primer duplex. (PDF 151 kb)

Supplementary Table 2

Nucleotide-composition analysis of T7 transcripts. (PDF 153 kb)

Supplementary Methods (PDF 312 kb)

Supplementary Discussion (PDF 25 kb)

Supplementary Data (PDF 837 kb)

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Hirao, I., Kimoto, M., Mitsui, T. et al. An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nat Methods 3, 729–735 (2006). https://doi.org/10.1038/nmeth915

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