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Construction of integrated gene logic-chip

Nature Nanotechnology volume 13, pages 933–940 (2018)Cite this article

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Abstract

In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.

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Fig. 1: Assembly and activity of the T7-chip.
Fig. 2: Properties and rational design of the gene nanochip activity.
Fig. 3: A single nanochip can express a defined gene in a (w/o) droplet
Fig. 4: Integration and reprogramming of the logic gate on the nanochip.
Fig. 5: Nanochip-based genetic circuit autonomously responds to the small RNA (miRNA) profile of a (w/o) droplet.

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Acknowledgements

The authors thank RIBM for AFM imaging, T. Sakurai, H. Okada and K. Nakamura (The University of Tokyo) for help with construction of the microfluidics system, and Y. Tomari and members of the Tomari Laboratory for providing comments on the miRNA studies and on this manuscript. The authors also thank Y. Hatano (Osaka University) and M. Hagiya (The University of Tokyo) for their comments on logic circuits, and I. Kawamata (Tohoku University) for comments on the kinetically based simulation of logic circuit. This work was partially supported by Grants-in-Aid for Scientific Research on Innovative Areas (‘Molecular Robotics’ to H.T. and M.E., nos. 15H00798 and 24104002; ‘Non-coding RNA Neo-taxonomy’ to H.T., no. 26113007), a Grant-in-Aid for Young Scientists (A), a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Challenging Exploratory Research (to H.T., nos. 24687018, 16KT0068 and 15K14485), Grants-in-Aid for Scientific Research (S) (to Y.H., no. 26220602) and (A) (to S.S., no. 16H02349), Grants-in-Aid for Young Scientists (B) (to R.I., no. 15K18668) from the Japan Ministry of Education, Culture, Sports, Science and Technology, Research Fellowships for Young Scientists (to T.M., no. 15J08491), Core-to-Core Program, A, Advanced Research Networks (Phototheranostics) (to R.I.) from the Japan Society for the Promotion of Science, CREST (to Y.H., no. JPMJCR1333), the Centre of Innovation (COI) Program (to T.F.) from the Japan Science and Technology Agency, the Cooperative Research Program of the Institute for Protein Research, Osaka University (CRa-18-01, to H.T and M.E.), the Asahi Glass Foundation, Futaba Electronics Memorial Foundation, and Hamaguchi Foundation for the Advancement of Biochemistry (to H.T.), and Futaba Electronics Memorial Foundation Scholarship (to T.M.).

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Author notes

  1. Takeya Masubuchi

    Present address: Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan

Authors and Affiliations

  1. Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan

    Takeya Masubuchi, Hao Qi, Ryosuke Iinuma, Yuya Miyazono, Takuya Ueda & Hisashi Tadakuma

  2. Graduate School of Science, Department of Chemistry, Kyoto University, Kyoto, Japan

    Masayuki Endo & Hiroshi Sugiyama

  3. Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan

    Masayuki Endo, Hiroshi Sugiyama, Yoshie Harada & Hisashi Tadakuma

  4. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Ryo Iizuka & Takashi Funatsu

  5. Department of Nanoscience and Nanoengineering (ASE Graduate School), Waseda University, Tokyo, Japan

    Ayaka Iguchi & Shuichi Shoji

  6. Research Organization for Nano & Life Innovation, Waseda University, Tokyo, Japan

    Dong Hyun Yoon & Tetsushi Sekiguchi

  7. Department of Chemical Engineering and Technology, Tianjin University, Tianjin, China

    Hao Qi

  8. Institute for Protein Research, Osaka University, Osaka, Japan

    Yoshie Harada & Hisashi Tadakuma

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Contributions

H.T. conceived, designed and supervised the study. T.M. performed the biochemical and microfluidics experiments and analysed the data. H.T. performed the AFM imaging with the help of RIBM (Tsukuba, Japan). R.I., T.F., S.S., A.I., D.H.Y, and T.S. constructed the microfluidics system. T.U. supervised H.T. and T.M. All authors discussed the results. H.T., H.S., T.M. and M.E. wrote the manuscript.

Corresponding authors

Correspondence to Masayuki Endo, Hiroshi Sugiyama, Takuya Ueda or Hisashi Tadakuma.

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Competing interests

The authors have a pending patent application on the programmable gene expression method in this work.

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Supplementary Figures 1–30, Supplementary Tables 1–4, Supplementary Notes and Supplementary References

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Masubuchi, T., Endo, M., Iizuka, R. et al. Construction of integrated gene logic-chip. Nature Nanotech 13, 933–940 (2018). https://doi.org/10.1038/s41565-018-0202-3

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