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Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA

Author

Listed:
  • Hinako Kawabe

    (University of Washington)

  • Christopher A. Thomas

    (University of Washington)

  • Shuichi Hoshika

    (Foundation for Applied Molecular Evolution
    Firebird Biomolecular Sciences LLC)

  • Myong-Jung Kim

    (Foundation for Applied Molecular Evolution
    Firebird Biomolecular Sciences LLC)

  • Myong-Sang Kim

    (Firebird Biomolecular Sciences LLC)

  • Logan Miessner

    (University of Washington)

  • Nicholas Kaplan

    (University of Washington)

  • Jonathan M. Craig

    (University of Washington)

  • Jens H. Gundlach

    (University of Washington)

  • Andrew H. Laszlo

    (University of Washington)

  • Steven A. Benner

    (Foundation for Applied Molecular Evolution
    Firebird Biomolecular Sciences LLC)

  • Jorge A. Marchand

    (University of Washington
    University of Washington)

Abstract

The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we devise an enzymatic strategy for inserting a singular, orthogonal xenonucleic acid (XNA) base pair into standard DNA sequences using 2′-deoxy-xenonucleoside triphosphates as substrates. Integrating this strategy with combinatorial oligos generated on a chip, we construct libraries containing single XNA bases for parameterizing kmer basecalling models for commercially available nanopore sequencing. These elementary steps are combined to synthesize and sequence DNA containing 12 letters – the upper limit of what is accessible within the electroneutral, canonical base pairing framework. By introducing low-barrier synthesis and sequencing strategies, this work overcomes previous obstacles paving the way for making expanded alphabets widely accessible.

Suggested Citation

  • Hinako Kawabe & Christopher A. Thomas & Shuichi Hoshika & Myong-Jung Kim & Myong-Sang Kim & Logan Miessner & Nicholas Kaplan & Jonathan M. Craig & Jens H. Gundlach & Andrew H. Laszlo & Steven A. Benne, 2023. "Enzymatic synthesis and nanopore sequencing of 12-letter supernumerary DNA," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42406-z
    DOI: 10.1038/s41467-023-42406-z
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    References listed on IDEAS

    as
    1. Henry H. Lee & Reza Kalhor & Naveen Goela & Jean Bolot & George M. Church, 2019. "Terminator-free template-independent enzymatic DNA synthesis for digital information storage," Nature Communications, Nature, vol. 10(1), pages 1-12, December.
    2. Denis A. Malyshev & Kirandeep Dhami & Thomas Lavergne & Tingjian Chen & Nan Dai & Jeremy M. Foster & Ivan R. Corrêa & Floyd E. Romesberg, 2014. "A semi-synthetic organism with an expanded genetic alphabet," Nature, Nature, vol. 509(7500), pages 385-388, May.
    3. Jay Shendure & Shankar Balasubramanian & George M. Church & Walter Gilbert & Jane Rogers & Jeffery A. Schloss & Robert H. Waterston, 2017. "DNA sequencing at 40: past, present and future," Nature, Nature, vol. 550(7676), pages 345-353, October.
    Full references (including those not matched with items on IDEAS)

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