In Vivo Replication of Semi-Synthetic

RESEARCH HIGHLIGHTS Nature Reviews Genetics | AOP, published online 20 May 2014; doi:10.1038/nrg3759 NPG SYNTHETIC BIOLOGY In vivo replication of semi-synthetic DNA In a new groundbreaking study, scientists from The Scripps Research Institute have engineered a semi-synthetic organism that contains an unnatural base pair (UBP; d5SICS∙dNaM) and that can reproduce normally. This is the first time that DNA containing a synthetic base pair has been replicated successfully in vivo, and this work has potential implications for information storage in DNA and the re-engineering of cells for a range of applications. Malyshev et al. first optimized conditions to increase the extracellular stability of synthetic nucleobases so that the half-lives of both d5SICS and dNaM were increased to ~9 hours. For successful replication, the unnatural nucleobases must be present inside the cell, so the authors then transformed Escherichia coli to contain an algal nucleotide triphosphate transporter (NTT2), which imports d5SICS and dNaM through the cell membrane. They also transformed the cells with an engineered plasmid (pINF) in which the dA∙dT base pair at position 505 was replaced with a base pair between dNaM and an analogue of d5SICS, so that later measurements of d5SICS would confirm the in vivo replication of the plasmid. After 15 hours of growth (~24 doublings) in medium containing the synthetic nucleobases, pINF was recovered from the cells and digested into nucleosides. To measure the level of UBP retention, the ratios of nucleosides (as identified from liquid chromatography–tandem mass spectrometry) were compared, which indicated the presence of ~1 UBP per plasmid. Additionally, PCR and sequencing of the region verified the retention of the UBP in >95% of the E. coli population. Both of these methods independently demonstrate in vivo replication of the semi-synthetic plasmid (the error rate of which was comparable to that seen in some natural systems) and confirm that the UBP had not been excised by a DNA repair pathway, which shows that living cells can replicate with an expanded genetic code. In summary, this paper shows that the NTT2 transporter imports the man- made nucleotides efficiently into E. coli, and that the endogenous replication machinery can process the synthetic nucleobases to successfully replicate DNA that contains a UBP. Although each organism contained only a single synthetic base pair, and it is not yet known whether these synthetic organisms can make proteins from the new genetic information, this pioneering work provides a platform for expanding the genetic ‘alphabet’. By increasing the amount of information that can be stored in DNA, the re-engineering of cells to produce proteins with a substantially expanded range of amino acids might now be a possibility. Bryony Jones ORIGINAL RESEARCH PAPER Malyshev, D. A. et al. A semi-synthetic organism with an expanded genetic alphabet. Nature http://dx.doi.org/10.1038/nature13314 (2014) NATURE REVIEWS | GENETICS VOLUME 15 | JULY 2014 © 2014 Macmillan Publishers Limited. All rights reserved.

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