Letter | Published:

A semi-synthetic organism with an expanded genetic alphabet

Nature 509, 385–388 (15 May 2014) | Download Citation

Subjects

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

References

  1. 1.

    et al. Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet. Proc. Natl Acad. Sci. USA 109, 12005–12010 (2012)

  2. 2.

    , , & Amplification, mutation, and sequencing of a six-letter synthetic genetic system. J. Am. Chem. Soc. 133, 15105–15112 (2011)

  3. 3.

    et al. Highly specific unnatural base pair systems as a third base pair for PCR amplification. Nucleic Acids Res. 40, 2793–2806 (2012)

  4. 4.

    , , , & Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs. J. Am. Chem. Soc. 133, 19878–19888 (2011)

  5. 5.

    , & Transcription of an expanded genetic alphabet. J. Am. Chem. Soc. 131, 5046–5047 (2009)

  6. 6.

    et al. Structural insights into DNA replication without hydrogen bonds. J. Am. Chem. Soc. 135, 18637–18643 (2013)

  7. 7.

    et al. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson–Crick geometry. Nature Chem. Biol. 8, 612–614 (2012)

  8. 8.

    , , , & Enzymatic phosphorylation of unnatural nucleosides. J. Am. Chem. Soc. 124, 14626–14630 (2002)

  9. 9.

    & Nucleoside monophosphate kinases: structure, mechanism, and substrate specificity. Adv. Enzymol. 73, 103–134 (1999)

  10. 10.

    & Non-mitochondrial ATP transport. Trends Biochem. Sci. 24, 64–68 (1999)

  11. 11.

    , & Deep origin of plastid/parasite ATP/ADP translocases. J. Mol. Evol. 56, 137–150 (2003)

  12. 12.

    , & Adenine nucleotide and lysine transport in Chlamydia psittaci. J. Bacteriol. 150, 662–670 (1982)

  13. 13.

    Rickettsial permeability: an ADP-ATP transport system. J. Biol. Chem. 251, 389–396 (1976)

  14. 14.

    & Bacterial endosymbionts of free-living amoebae. J. Eukaryot. Microbiol. 51, 509–514 (2004)

  15. 15.

    et al. Tapping the nucleotide pool of the host: novel nucleotide carrier proteins of Protochlamydia amoebophila. Mol. Microbiol. 60, 1534–1545 (2006)

  16. 16.

    & Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298 (1996)

  17. 17.

    & Functional expression and characterisation of membrane transport proteins. Plant Biol. 14, 675–690 (2012)

  18. 18.

    et al. Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl Acad. Sci. USA 106, 3621–3626 (2009)

  19. 19.

    et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006)

  20. 20.

    , & Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs. Chemistry 18, 1231–1239 (2012)

  21. 21.

    , , & Optimization of an unnatural base pair toward natural-like replication. J. Am. Chem. Soc. 131, 3246–3252 (2009)

  22. 22.

    et al. Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications. J. Am. Chem. Soc. 136, 826–829 (2014)

  23. 23.

    & Initiation of DNA synthesis in Escherichia coli. Annu. Rev. Biochem. 48, 999–1034 (1979)

  24. 24.

    et al. Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication. Nucleic Acids Res. 39, 7020–7033 (2011)

  25. 25.

    et al. Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA. Nature 506, 391–395 (2013)

  26. 26.

    , , & PCR with an expanded genetic alphabet. J. Am. Chem. Soc. 131, 14620–14621 (2009)

  27. 27.

    et al. An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA. Nature Methods 3, 729–735 (2006)

  28. 28.

    Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71, 17–50 (2002)

  29. 29.

    & Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nature Protocols 6, 242–251 (2011)

  30. 30.

    & Rapid and efficient synthesis of nucleoside 5′-0-(1-thiotriphosphates), 5′-triphosphates and 2',3′-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one. J. Org. Chem. 54, 631–635 (1989)

  31. 31.

    & Precipitation of Large, High-Abundance Proteins from Serum with Organic Solvents in ABRF 2003: Translating Biology using Proteomics and Functional Genomics Poster no. P111-W (2003)

  32. 32.

    & Determination of bacterial cell volume with the Coulter Counter. J. Bacteriol. 168, 1466–1467 (1986)

  33. 33.

    , , & Expanding coverage of the metabolome for global metabolite profiling. Anal. Chem. 83, 2152–2161 (2011)

  34. 34.

    , , & Introduction of plasmid DNA into cells. Curr. Prot. Mol. Biol Chapter 1, Unit 1.8 (2001)

  35. 35.

    , , , & Nucleotide parasitism by Simkania negevensis (Chlamydiae). J. Bacteriol. 193, 225–235 (2011)

  36. 36.

    & Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides. J. Bacteriol. 188, 6261–6268 (2006)

  37. 37.

    , & Allosteric regulation of Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo. J. Biol. Chem. 273, 34098–34104 (1998)

  38. 38.

    , & Comparison of methanol and perchloric acid extraction procedures for analysis of nucleotides by isotachophoresis. J. Chromatogr. B Biomed. Sci. Appl. 374, 162–169 (1986)

Download references

Acknowledgements

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

Affiliations

  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

Authors

  1. Search for Denis A. Malyshev in:

  2. Search for Kirandeep Dhami in:

  3. Search for Thomas Lavergne in:

  4. Search for Tingjian Chen in:

  5. Search for Nan Dai in:

  6. Search for Jeremy M. Foster in:

  7. Search for Ivan R. Corrêa in:

  8. Search for Floyd E. Romesberg in:

Contributions

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

Extended data figures

  1. 1.

    Natural triphosphate uptake by NTTs.

  2. 2.

    Degradation of unnatural triphosphates in growth media.

  3. 3.

    Effect of potassium phosphate on dATP uptake and stability in growth media.

  4. 4.

    dATP uptake and growth of cells expressing PtNTT2 as a function of inducer (IPTG) concentration.

  5. 5.

    Stability and uptake of dATP in the presence of 50 mM KPi and 1 mM IPTG.

  6. 6.

    Calibration of the streptavidin shift (SAS).

  7. 7.

    Decomposition of unnatural triphosphates, pINF quantification, and retention of the UBP with extended cell growth.

Extended data tables

  1. 1.

    OD600 of E. coli cultures and relative copy number of plasmid (pINF or control pUC19) as determined by its molar ratio to pACS after 19 h of growth

  2. 2.

    Relative quantification by LC-MS/MS using synthetic oligonucleotides containing d5SICS and dNaM

  3. 3.

    Summary of the most successful extraction methods

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.

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13314

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.