Abstract

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of 200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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Acknowledgements

We thank S. Sehgal, A.K. Sreekumar, E. Baidoo, C.J. Petzold, L. Chan, and V. Teixeira Benites for assistance with experiments, G. Goyal, J. Chiniquy, and N. Kaplan for NGS of the synthesis products, C. Hoover for optimizing the NGS procedure, P.D. Adams, C.J. Joshua, J.F. Barajas, M.E. Brown, C.B. Eiben, A. Flamholz, A. Tambe, B. Wagner, S. Weißgraeber, P. Weißgraeber, S. Jager, and R. Palluk for helpful discussions, and E. de Ugarte for assistance with artwork. This work has been supported by the DOE Joint BioEnergy Institute (https://www.jbei.org) by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. D.H.A. was also supported by the Synthetic Biology Engineering Research Center (SynBERC) through National Science Foundation grant NSF EEC 0540879 and by NIH training grant GM-08295 through NIGMS. T.d.R. was supported by ERASynBio (81861: “SynPath”). N.J.H. was also supported by the DOE Joint Genome Institute (https://jgi.doe.gov) by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy's National Nuclear Security Administration under contract DE-NA0003525. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains, a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).

Author information

Author notes

    • Sebastian Palluk
    •  & Daniel H Arlow

    These authors contributed equally to this work.

Affiliations

  1. Joint BioEnergy Institute, Emeryville, California, USA.

    • Sebastian Palluk
    • , Daniel H Arlow
    • , Tristan de Rond
    • , Sebastian Barthel
    • , Justine S Kang
    • , Rathin Bector
    • , Hratch M Baghdassarian
    • , Alisa N Truong
    • , Peter W Kim
    • , Anup K Singh
    • , Nathan J Hillson
    •  & Jay D Keasling
  2. Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA.

    • Sebastian Palluk
    • , Daniel H Arlow
    • , Tristan de Rond
    • , Sebastian Barthel
    • , Justine S Kang
    • , Rathin Bector
    • , Hratch M Baghdassarian
    • , Alisa N Truong
    • , Nathan J Hillson
    •  & Jay D Keasling
  3. Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.

    • Sebastian Palluk
    •  & Sebastian Barthel
  4. Biophysics Graduate Group, UC Berkeley, Berkeley, California, USA.

    • Daniel H Arlow
  5. Institute for Quantitative Biosciences, UC Berkeley, Berkeley, California, USA.

    • Daniel H Arlow
    •  & Jay D Keasling
  6. Department of Chemistry, UC Berkeley, Berkeley, California, USA.

    • Tristan de Rond
  7. Department of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California, USA.

    • Justine S Kang
    • , Rathin Bector
    •  & Jay D Keasling
  8. Department of Bioengineering UC Berkeley, Berkeley, California, USA.

    • Hratch M Baghdassarian
    •  & Jay D Keasling
  9. CBRN Defense and Energy Technologies, Sandia National Laboratories, Livermore, California, USA.

    • Peter W Kim
    •  & Anup K Singh
  10. DOE Joint Genome Institute, Walnut Creek, California, USA.

    • Nathan J Hillson
  11. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.

    • Jay D Keasling

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Contributions

S.P. and D.H.A. conceived the method, designed and performed experiments, analyzed data, and wrote the manuscript with input from all other authors. T.d.R., S.B., J.S.K., R.B., H.M.B., A.N.T., and P.W.K. performed experiments and analyzed data, and all authors discussed and interpreted results. A.K.S., N.J.H. and J.D.K. supervised the research. All authors read and corrected the manuscript.

Competing interests

S.P. and D.H.A. have filed international patent application WO2017223517A1 on polymerase-nucleotide conjugates and are cofounders of Ansa Biotechnologies, Inc.

Corresponding authors

Correspondence to Daniel H Arlow or Jay D Keasling.

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https://doi.org/10.1038/nbt.4173