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Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription

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Abstract

A phosphoramidite monomer bearing an azobenzene is synthesized from D-threoninol. Using this monomer, azobenzene moieties can be introduced into oligodeoxyribonucleotide (DNA) at any position on a conventional DNA synthesizer. With this azobenzene-tethered DNA, formation and dissociation of a DNA duplex can be reversibly photo-regulated by cis–trans isomerization of the azobenzene. When the azobenzene takes a trans-form, a stable duplex is formed. After isomerization of the trans-azobenzene to its cis-form by UV-light irradiation (300 nm < λ < 400 nm), the duplex can be dissociated into two strands. The duplex is reformed on photo-induced cis–trans isomerization (λ > 400 nm). The introduction of azobenzenes into the T7 promoter at specific positions also efficiently and reversibly photo-regulates transcription by T7-RNA polymerase. The reversible regulation can be repeated many times without causing damage to the DNA or the azobenzene moiety. These procedures take approximately 10 d to complete.

NOTE: In Figure 4 of the version of this article originally published online, the base sequence of the oligonucleotide was incorrect. The figure has been replaced in all versions of the article.

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Figure 1: Synthesis of a modified DNA sequence tethering an azobenzene moiety (X residue)8.
Figure 2: Sequence design of the azobenzene-tethered DNA for effective photo-regulation.
Figure 3: Schematic representation.
Figure 4: Schematic representation.
Figure 5: Fluorescence spectra of single-stranded FAM-DNA (green line) and FAM-DNA/Dabcyl-DNA duplex (black, red and blue lines) at 37 °C by excitation at 480 nm.
Figure 6: Photo-switching of transcription reaction by T7-RNA polymerase with the photo-responsive T7 promoter by irradiating with either visible light (trans-form) or UV light (cis-form).

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  • 01 March 2007

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Acknowledgements

This work was supported by Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST). Partial support through a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and The Mitsubishi Foundation (for H.A.) is also acknowledged.

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Authors and Affiliations

  1. Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan

    Hiroyuki Asanuma, Xingguo Liang & Hidenori Nishioka

  2. CREST, Japan Science and Technology Agency (JST), Kawaguchi, 332-0012, Japan

    Hiroyuki Asanuma

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

    Daijiro Matsunaga, Mingzhe Liu & Makoto Komiyama

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  1. Hiroyuki Asanuma
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  2. Xingguo Liang
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  3. Hidenori Nishioka
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  4. Daijiro Matsunaga
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  5. Mingzhe Liu
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Correspondence to Hiroyuki Asanuma or Makoto Komiyama.

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Asanuma, H., Liang, X., Nishioka, H. et al. Synthesis of azobenzene-tethered DNA for reversible photo-regulation of DNA functions: hybridization and transcription. Nat Protoc 2, 203–212 (2007). https://doi.org/10.1038/nprot.2006.465

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