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Autonomous and Programmable Strand Generator Implemented as DNA and Enzymatic Chemical Reaction Cascade

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Abstract

Nanomechanical devices are driven by chemical and/or physical signals, which exert their functions by transitioning between different molecular states. Many DNA-based nanomechanical devices proposed so far are typically driven by applying single-stranded DNAs representing instructions. To use these devices, it is necessary to manually deliver the strands in a step-by-step manner or use an external computerized apparatus to supply the strands. In this work, we propose an elaborate DNA reaction system capable of generating a sequence of multiple single-stranded DNAs according to a given program which is also a single-stranded DNA. The system is designed as a cascade of DNA hybridization and enzymatic reactions, in which the whole reaction is triggered by the program strand. The system can generate single-stranded DNAs one by one in a sequential order written on the program strand. We experimentally demonstrated the feasibility and the scalability of the system, by using the program strands encoding instruction sequences of different lengths.

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Acknowledgements

The authors appreciate Shawn Douglas for holding BIOMOD competition where we began this research. We thank the member of BIOMOD team of Tohoku University to come up with the first preliminary idea of the project, who are Shota Kawakami, Yu-chin Chen, Shogo Hiratsuka, Sho Aradachi, Daisuke Tamatsuki, Hayato Yuuki, Takuto Takahashi, Eiki Ishihara, Shunsuke Imai, Hayato Otaka, Yuto Otaki, Kenta Suzuki, Taiki Watanabe, Fumi Takabatake. We also thank Kaori Tanabe for helping the experiment. This work was supported by JSPS KAKENHI Grant numbers 15H01715, 18K18144, 19KK0261, 20H00618, 20H00619, 20H05701, 20H05968, 20H05969, 20H05970, 20H05971, 20K20979, and the 51st Research Grants in the Natural Sciences of Mitsubishi Foundation.

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Authors and Affiliations

  1. Department of Robotics, Graduate School of Engineering, Tohoku University, 6-6-01, Aramaki Aza Aoba, Aoba, Sendai, Miyagi, 980-8579, Japan

    Ibuki Kawamata, Shin-ichiro M. Nomura & Satoshi Murata

  2. Natural Science Division, Faculty of Core Research, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo, 112-8610, Japan

    Ibuki Kawamata

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  1. Ibuki Kawamata
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  2. Shin-ichiro M. Nomura
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  3. Satoshi Murata
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Correspondence to Ibuki Kawamata.

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Appendices

Appendix A: Experiment

All DNAs were purchased from Eurofins Genomics Japan. The sequences of DNA are listed in the Table 1. The concentration of DNA in the initial state was 200 nM for all experiments, unless otherwise specified. To stabilize the DNA complex, all the DNA samples were annealed from 95 to 20 \({}^\circ \)C by a temperature ramp of \(-1~{}^\circ \)C per 1 min. For enzymes, we used Bst polymerase (New England Biolabs), Nt.BstNBI (New England Biolabs) for nickase, and EcoRI (Takara, Japan) restriction enzyme for endonuclease activity. The concentration of enzymes were 80, 200, 150 U/mL for polymerase, nickase and restriction enzyme, respectively. The concentration of dNTP was 0.8 mM. All reactions were carried at 45 \(^\circ \)C in H buffer (TAKARA BIO). After an incubation of prescribed time, the reactions were stopped by inactivating enzymes at 80 \(^\circ \)C for 10 min, followed by a gradual annealing from 95 to 25 \(^\circ \)C by \(-1~{}^\circ \) per 1 min. The gel was imaged by ChemiDoc (BIO-RAD) and analyzed by ImageLab (BIORAD) and ImageJ (NIH, USA).

Appendix B: Sequence of DNA

Sequences of DNAs are listed in the following table. The 3\(^{\prime }\)-ends of template, output, transducer, gate, update, and adjuster were phosphorylated, unless otherwise specified.

Table 1 Sequences of DNAs
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Appendix C: Raw Gel Electrophoresis Data

Details of step-by-step experiments are summarized in Fig. 5. Although Bst polymerase, Nt.BstNBI, and EcoRI have recommended reaction temperatures of 65,  65, and 37 \(^\circ \)C, respectively, all of the enzymes show activity under 45 \(^\circ \)C.

Fig. 5
figure 5

Gel electrophoresis images that show the reaction of each process. Each image corresponds to the reactions a from 2 to 4, b from 5 to 6, c from 7 to 8, and d from 10 to 13. DNA strands in a, c, and d were stained by SYBR GOLD, while fluorescence of TAMRA which was modified to the 3\(^{\prime }\)-end of output is detected in b. The presence and absence of corresponding DNA structure or enzyme is represented by plus and minus

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The result of a single-instruction system is shown in Fig. 6.

Fig. 6
figure 6

Gel electrophoresis of single-instruction system

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The effect of phosphorylations is summarized in Fig. 7.

Fig. 7
figure 7

Experimental results of two instruction input, using phosphorylated DNA

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The raw gel electrophoresis data for the three instruction systems are summarized in Fig. 8, which are used to make the graphs of Fig. 3.

Fig. 8
figure 8

Raw gel electrophoresis data for three instruction system

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Kawamata, I., Nomura, Si.M. & Murata, S. Autonomous and Programmable Strand Generator Implemented as DNA and Enzymatic Chemical Reaction Cascade. New Gener. Comput. 40, 723–736 (2022). https://doi.org/10.1007/s00354-022-00156-4

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  • DOI: https://doi.org/10.1007/s00354-022-00156-4

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