A semi-synthetic organism with an expanded genetic alphabet

We have developed a class of unnatural base pairs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM, which is efficiently PCR amplified1 and transcribed4,5 in vitro, and whose unique mechanism of replication has been characterized6,7

Denis A. Malyshev

2014

Scholarcy highlights

  • Organisms are defined by the information encoded in their genomes, and since the evolution of life, this information has been encoded using a two base pair genetic alphabet
  • To make the unnatural triphosphates available inside the cell, we previously suggested using passive diffusion of the free nucleosides into the cytoplasm followed by their conversion to the corresponding triphosphate via the nucleoside salvage pathway
  • Transport via an nucleotide triphosphate transporters requires that the unnatural triphosphates are sufficiently stable in culture media; preliminary characterization of d5SICSTP and dNaMTP indicated that decomposition occurs in the presence of actively growing E. coli
  • Cells expressing PtNTT2 were found to have the highest levels of intracellular-dATP, and while both extra- and intercellular dephosphorylation was still observed, the ratio of triphosphate to dephosphorylation products inside the cell remained roughly constant, indicating that the extracellular concentrations and PtNTT2-mediated influx are sufficient to compensate for intracellular decomposition
  • We found that the addition of KPi increased the extracellular stability of d5SICSTP and dNaMTP, and when a stationary phase culture was diluted 100-fold into fresh media, the half-lives of both unnatural triphosphates were found to be ~9 h, which seemed sufficient for our purposes
  • We have demonstrated that PtNTT2 efficiently imports d5SICSTP and dNaMTP into E. coli by PtNTT2 and that an E. coli polymerase, possibly Polymerase I, is able to efficiently use the unnatural triphosphates to replicate DNA containing the unnatural base pairs within the cellular environment with reasonable efficiency and fidelity
  • Fragment generation for sequencing—Purified mixtures of pINF and pACS plasmids after the overnight growth were amplified by PCR under the following conditions: 1× OneTaq reaction buffer, MgSO4 adjusted to 3.0 mM, 0.2 mM of dNTP, 0.1 mM of dNaMTP, 0.1 mM of the d5SICSTP analog dTPT3TP, 1 μM of each of the primers pUC19seq2-fwd and pUC19-seq-rev, and 0.02 U/μl of OneTaq DNA Polymerase in a total volume of 25 μl under the following thermal cycling conditions: denaturation; and 10 cycles of denaturation, and extension 68 °C, 2 min

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