A semi-synthetic organism with an expanded genetic alphabet

  • Nature volume 509, pages 385388 (15 May 2014)
  • doi:10.1038/nature13314
  • Download Citation


Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A–T and G–C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs)1,2,3. We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM), which is efficiently PCR-amplified1 and transcribed4,5 in vitro, and whose unique mechanism of replication has been characterized6,7. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS–dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.

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We thank I. Haferkamp and J. Audia for kindly providing the NTT plasmids and helpful discussions, and P. Ordoukhanian for providing access to the Center for Protein and Nucleic Acid Research at TSRI. This work was supported by the US National Institutes of Health (NIH) (GM 060005).

Author information


  1. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Denis A. Malyshev
    • , Kirandeep Dhami
    • , Thomas Lavergne
    • , Tingjian Chen
    •  & Floyd E. Romesberg
  2. New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA

    • Nan Dai
    • , Jeremy M. Foster
    •  & Ivan R. Corrêa


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D.A.M., K.D., T.C. and F.E.R. designed the experiments. D.A.M., K.D. and T.L. performed the experiments. N.D., J.M.F. and I.R.C.J. performed LC-MS/MS analysis. D.A.M., K.D. and F.E.R. analysed data and D.A.M. and F.E.R. wrote the manuscript with assistance from the other authors.

Competing interests

F.E.R. and D.A.M. have filed a patent application based on the use of NTTs for biotechnological applications. F.E.R. D.A.M., T.L. and K.D. have shares in Synthorx Inc., a company that has commercial interests in the UBP. D.A.M. and K.D. are currently employed by Synthorx Inc. The other authors declare no competing financial interests.

Corresponding author

Correspondence to Floyd E. Romesberg.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    The file contains the sequences of oligonucleotides used in this study, an example calculation of plasmid amplification, and the sequence of the pACS plasmid.

Excel files

  1. 1.

    Supplementary Data

    This file contains raw sequencing traces for PCR fragments generated from pINF plasmid propagated in E. coli at different time points (n=3). The position of the unnatural nucleotide is indicated with a red arrow.


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