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. 2014 May 15;509(7500):385-8.
doi: 10.1038/nature13314. Epub 2014 May 7.

A Semi-Synthetic Organism With an Expanded Genetic Alphabet

Free PMC article

A Semi-Synthetic Organism With an Expanded Genetic Alphabet

Denis A Malyshev et al. Nature. .
Free PMC article


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). 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-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. 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.


Figure 1
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 total intracellular concentration of triphosphate. Errors represent s.d. of the mean, n=3.
Figure 2
Figure 2. Intracellular UBP replication
a, Structure of pACS and pINF. dX and dY correspond to dNaM and a d5SICS analog that facilitated plasmid construction (see Methods). cloDF = origin of replication; Sm = streptomycin resistance gene; AmpR = ampicillin resistance gene; ori = ColE1 origin of replication; MCS = multiple cloning site; 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 PCR. X = dNaM, Y’ = dTPT3 (an analog of d5SICS22), and Y = d5SICS (see text). Color indicates regions of homology. The doubly-nicked product was used directly to transform E. coli harboring pACS. c, The addition of d5SICSTP and dNaMTP eliminates a growth lag of cells harboring pINF. EP=electroporation. Errors 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× 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 nt region containing the site of UBP incorporation (nt 437–630) was amplified and biotinylated. B=biotin; SA=streptavidin. The natural pUC19 control plasmid was prepared identically to pINF. 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).
Figure 3
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 (blue squares), OD600 (black diamonds), and relative amount of d5SICSTP and dNaMTP in the media (red and green circles, respectively; 100% = 0.25 mM), were determined as a function of time. Errors represent s.d. of the mean, n=3.

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    1. Malyshev DA, 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. 2012;109:12005–12010. - PMC - PubMed
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