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Nature | Letter

日本語要約

A semi-synthetic organism with an expanded genetic alphabet

Journal name:
Nature
Volume:
509,
Pages:
385–388
Date published:
DOI:
doi:10.1038/nature13314
Received
Accepted
Published online

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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Figures

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  1. Nucleoside triphosphate stability and import.
    Figure 1: Nucleoside triphosphate stability and import.

    a, Chemical structure of the d5SICS–dNaM UBP compared to the natural dG–dC base pair. b, Composition analysis of d5SICS and dNaM in the media (top) and cytoplasmic (bottom) fractions of cells expressing PtNTT2 after 30 min incubation; dA shown for comparison. 3P, 2P, 1P and 0P correspond to triphosphate, diphosphate, monophosphate and nucleoside, respectively; [3P] is the intracellular concentration of triphosphate. Error bars represent s.d. of the mean, n = 3.

  2. Intracellular UBP replication.
    Figure 2: Intracellular UBP replication.

    a, Structure of pACS and pINF. dX and dY correspond to dNaM and a d5SICS analogue22 that facilitated plasmid construction (see Methods). cloDF, origin of replication; Sm, streptomycin resistance gene; AmpR, ampicillin resistance gene; ori, ColE1 origin of replication; lacZα, β-galactosidase fragment gene. b, Overview of pINF construction. A DNA fragment containing the unnatural nucleotide was synthesized via solid-phase DNA synthesis and then used to assemble synthetic pINF via circular-extension PCR29. X, dNaM; Y′, dTPT3 (an analogue of d5SICS22); y, d5SICS (see text). Colour indicates regions of homology. The doubly nicked product was used directly to transform E. coli harbouring pACS. c, The addition of d5SICSTP and dNaMTP eliminates a growth lag of cells harbouring pINF. EP, electroporation. Error bars represent s.d. of the mean, n = 3. d, LC-MS/MS total ion chromatogram of global nucleoside content in pINF and pUC19 recorded in dynamic multiple reaction monitoring (DMRM) mode. pINF and pUC19 (control) were propagated in E. coli in the presence or absence of unnatural triphosphates, and with or without PtNTT2 induction. The inset shows a 100-fold expansion of the mass-count axis in the d5SICS region. e, Biotinylation only occurs in the presence of the UBP, the unnatural triphosphates and transporter induction. After growth, pINF was recovered, and a 194-nucleotide region containing the site of UBP incorporation (nucleotides 437–630) was amplified and biotinylated. B, biotin; SA, streptavidin. The natural pUC19 control plasmid was prepared identically to pINF. A 50-bp DNA ladder is shown to the left. f, Sequencing analysis demonstrates retention of the UBP. An abrupt termination in the Sanger sequencing reaction indicates the presence of UBP incorporation (site indicated with arrow).

  3. Intracellular stability of the UBP.
    Figure 3: Intracellular stability of the UBP.

    E. coli C41(DE3) pACS was transformed with pINF and grown after a single dose of d5SICSTP and dNaMTP was provided in the media. UBP retention in recovered pINF, OD600, and relative amount of d5SICSTP and dNaMTP in the media (100% = 0.25 mM), were determined as a function of time. Error bars represent s.d. of the mean, n = 3.

  4. Natural triphosphate uptake by NTTs.
    Extended Data Fig. 1: Natural triphosphate uptake by NTTs.

    a, Survey of reported substrate specificity (KM, μM) of the NTTs assayed in this study. b, PtNTT2 is significantly more active in the uptake of [α-32P]-dATP compared to other nucleotide transporters. Raw (left) and processed (right) data are shown. Relative radioactivity corresponds to the total number of counts produced by each sample. Interestingly, both PamNTT2 and PamNTT5 exhibit a measurable uptake of dATP although this activity was not reported before. This can possibly be explained by the fact that substrate specificity was only characterized using competition experiments, and assay sensitivity might not have been adequate to detect this activity15. References 35, 36 are cited in this figure.

  5. Degradation of unnatural triphosphates in growth media.
    Extended Data Fig. 2: Degradation of unnatural triphosphates in growth media.

    Unnatural triphosphates (3P) of dNaM and d5SICS are degraded to diphosphates (2P), monophosphates (1P) and nucleosides (0P) in the growing bacterial culture. Potassium phosphate (KPi) significantly slows down the dephosphorylation of both unnatural triphosphates. a, Representative HPLC traces (for the region between ~20 and 24 min). dNaM and d5SICS nucleosides are eluted at approximately 40 min and not shown. b, Composition profiles.

  6. Effect of potassium phosphate on dATP uptake and stability in growth media.
    Extended Data Fig. 3: Effect of potassium phosphate on dATP uptake and stability in growth media.

    a, KPi inhibits the uptake of [α-32P]-dATP at concentrations above 100 mM. Raw (left) and processed (right) data are shown. The NTT from Rickettsia prowazekii (RpNTT2) does not mediate the uptake of any of the dNTPs and was used as a negative control: its background signal was subtracted from those of PtNTT2 (black bars) and TpNTT2 (white bars). Relative radioactivity corresponds to the total number of counts produced by each sample. b, KPi (50 mM) significantly stabilizes [α-32P]-dATP in the media. Triphosphate stability in the media is not significantly affected by the nature of the NTT expressed. 3P, 2P and 1P correspond to triphosphate, diphosphate and monophosphate states, respectively. Error bars represent s.d. of the mean, n = 3.

  7. dATP uptake and growth of cells expressing PtNTT2 as a function of inducer (IPTG) concentration.
    Extended Data Fig. 4: dATP uptake and growth of cells expressing PtNTT2 as a function of inducer (IPTG) concentration.

    Growth curves and [α-32P]-dATP uptake by bacterial cells transformed with pCDF-1b-PtNTT2 (pACS) plasmid as a function of IPTG concentration. a, Total uptake of radioactive substrate (left) and total intracellular triphosphate content (right) are shown at two different time points. Relative radioactivity corresponds to the total number of counts produced by each sample. b, A stationary phase culture of C41(DE3) pACS cells was diluted 100-fold into fresh 2 × YT media containing 50 mM KPi, streptomycin, and IPTG at the indicated concentrations and were grown at 37°C. Error bars represent s.d. of the mean, n = 3.

  8. Stability and uptake of dATP in the presence of 50 mM KPi and 1 mM IPTG.
    Extended Data Fig. 5: Stability and uptake of dATP in the presence of 50 mM KPi and 1 mM IPTG.

    Composition of [α-32P]-dATP in the media (left) and cytoplasmic fraction (right) as a function of time. TLC images and their quantifications are shown at the bottom and the top of each of the panels, respectively. 3P, 2P and 1P correspond to nucleoside triphosphate, diphosphate and monophosphate, respectively. M refers to a mixture of all three compounds that was used as a TLC standard. The position labelled ‘Start’ corresponds to the position of sample spotting on the TLC plate.

  9. Calibration of the streptavidin shift (SAS).
    Extended Data Fig. 6: Calibration of the streptavidin shift (SAS).

    a, The SAS is plotted as a function of the fraction of template containing the UBP. Error bars represent s.d. of the mean, n = 3. b, Representative data. SA, streptavidin.

  10. Decomposition of unnatural triphosphates, pINF quantification, and retention of the UBP with extended cell growth.
    Extended Data Fig. 7: Decomposition of unnatural triphosphates, pINF quantification, and retention of the UBP with extended cell growth.

    a, Dephosphorylation of the unnatural nucleoside triphosphate. 3P, 2P, 1P and 0P correspond to triphosphate, diphosphate, monophosphate and nucleoside states, respectively. The composition at the end of the 1 h recovery is shown at the right. b, Restriction analysis of pINF and pACS plasmids purified from E. coli, linearized with NdeI restriction endonuclease and separated on a 1% agarose gel (assembled from independent gel images). Molar ratios of pINF/pACS plasmids are shown at the top of each lane. For each time point, triplicate data are shown in three lanes with the untransformed control shown in the fourth, rightmost lane (see Methods). c, Number of pINF doublings as a function of time. The decrease starting at approximately 50 h is due to the loss of the pINF plasmid that also results in increased error. See the section on pINF replication in E. coli in the Methods for details. d, UBP retention (%) as a function of growth as determined by gel shift (data shown in Fig. 3) and Sanger sequencing (sequencing traces are available as Supplementary Data). In a, c and d, error shown is the s.d. of mean, n = 3.

Tables

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  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
    Extended Data Table 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. Relative quantification by LC-MS/MS using synthetic oligonucleotides containing d5SICS and dNaM
    Extended Data Table 2: Relative quantification by LC-MS/MS using synthetic oligonucleotides containing d5SICS and dNaM
  3. Summary of the most successful extraction methods
    Extended Data Table 3: Summary of the most successful extraction methods

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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

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 financial 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.

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Natural triphosphate uptake by NTTs. (65 KB)

    a, Survey of reported substrate specificity (KM, μM) of the NTTs assayed in this study. b, PtNTT2 is significantly more active in the uptake of [α-32P]-dATP compared to other nucleotide transporters. Raw (left) and processed (right) data are shown. Relative radioactivity corresponds to the total number of counts produced by each sample. Interestingly, both PamNTT2 and PamNTT5 exhibit a measurable uptake of dATP although this activity was not reported before. This can possibly be explained by the fact that substrate specificity was only characterized using competition experiments, and assay sensitivity might not have been adequate to detect this activity15. References 35, 36 are cited in this figure.

  2. Extended Data Figure 2: Degradation of unnatural triphosphates in growth media. (86 KB)

    Unnatural triphosphates (3P) of dNaM and d5SICS are degraded to diphosphates (2P), monophosphates (1P) and nucleosides (0P) in the growing bacterial culture. Potassium phosphate (KPi) significantly slows down the dephosphorylation of both unnatural triphosphates. a, Representative HPLC traces (for the region between ~20 and 24 min). dNaM and d5SICS nucleosides are eluted at approximately 40 min and not shown. b, Composition profiles.

  3. Extended Data Figure 3: Effect of potassium phosphate on dATP uptake and stability in growth media. (210 KB)

    a, KPi inhibits the uptake of [α-32P]-dATP at concentrations above 100 mM. Raw (left) and processed (right) data are shown. The NTT from Rickettsia prowazekii (RpNTT2) does not mediate the uptake of any of the dNTPs and was used as a negative control: its background signal was subtracted from those of PtNTT2 (black bars) and TpNTT2 (white bars). Relative radioactivity corresponds to the total number of counts produced by each sample. b, KPi (50 mM) significantly stabilizes [α-32P]-dATP in the media. Triphosphate stability in the media is not significantly affected by the nature of the NTT expressed. 3P, 2P and 1P correspond to triphosphate, diphosphate and monophosphate states, respectively. Error bars represent s.d. of the mean, n = 3.

  4. Extended Data Figure 4: dATP uptake and growth of cells expressing PtNTT2 as a function of inducer (IPTG) concentration. (64 KB)

    Growth curves and [α-32P]-dATP uptake by bacterial cells transformed with pCDF-1b-PtNTT2 (pACS) plasmid as a function of IPTG concentration. a, Total uptake of radioactive substrate (left) and total intracellular triphosphate content (right) are shown at two different time points. Relative radioactivity corresponds to the total number of counts produced by each sample. b, A stationary phase culture of C41(DE3) pACS cells was diluted 100-fold into fresh 2 × YT media containing 50 mM KPi, streptomycin, and IPTG at the indicated concentrations and were grown at 37°C. Error bars represent s.d. of the mean, n = 3.

  5. Extended Data Figure 5: Stability and uptake of dATP in the presence of 50 mM KPi and 1 mM IPTG. (196 KB)

    Composition of [α-32P]-dATP in the media (left) and cytoplasmic fraction (right) as a function of time. TLC images and their quantifications are shown at the bottom and the top of each of the panels, respectively. 3P, 2P and 1P correspond to nucleoside triphosphate, diphosphate and monophosphate, respectively. M refers to a mixture of all three compounds that was used as a TLC standard. The position labelled ‘Start’ corresponds to the position of sample spotting on the TLC plate.

  6. Extended Data Figure 6: Calibration of the streptavidin shift (SAS). (150 KB)

    a, The SAS is plotted as a function of the fraction of template containing the UBP. Error bars represent s.d. of the mean, n = 3. b, Representative data. SA, streptavidin.

  7. Extended Data Figure 7: Decomposition of unnatural triphosphates, pINF quantification, and retention of the UBP with extended cell growth. (185 KB)

    a, Dephosphorylation of the unnatural nucleoside triphosphate. 3P, 2P, 1P and 0P correspond to triphosphate, diphosphate, monophosphate and nucleoside states, respectively. The composition at the end of the 1 h recovery is shown at the right. b, Restriction analysis of pINF and pACS plasmids purified from E. coli, linearized with NdeI restriction endonuclease and separated on a 1% agarose gel (assembled from independent gel images). Molar ratios of pINF/pACS plasmids are shown at the top of each lane. For each time point, triplicate data are shown in three lanes with the untransformed control shown in the fourth, rightmost lane (see Methods). c, Number of pINF doublings as a function of time. The decrease starting at approximately 50 h is due to the loss of the pINF plasmid that also results in increased error. See the section on pINF replication in E. coli in the Methods for details. d, UBP retention (%) as a function of growth as determined by gel shift (data shown in Fig. 3) and Sanger sequencing (sequencing traces are available as Supplementary Data). In a, c and d, error shown is the s.d. of mean, n = 3.

Extended Data Tables

  1. Extended Data Table 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 (60 KB)
  2. Extended Data Table 2: Relative quantification by LC-MS/MS using synthetic oligonucleotides containing d5SICS and dNaM (89 KB)
  3. Extended Data Table 3: Summary of the most successful extraction methods (84 KB)

Supplementary information

PDF files

  1. Supplementary Information (243 KB)

    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. Supplementary Data (363 KB)

    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.

Additional data