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jueves, 8 de mayo de 2014

A semi-synthetic organism with an expanded genetic alphabet

Triphosphates of hydrophobic nucleotides d5SICS and dNaM are imported into Escherichia coli by an exogenous algal nucleotide triphosphate transporter and then used by an endogenous polymerase to replicate, and faithfully maintain over many generations of growth, a plasmid containing the d5SICS–dNaM unnatural base pair. 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 alphabetNucleoside 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.

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