Dataset Information

Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

ABSTRACT: Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA containing dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.

SUBMITTER: Ledbetter MP

PROVIDER: S-EPMC5793209 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

Ledbetter Michael P MP Karadeema Rebekah J RJ Romesberg Floyd E FE

Journal of the American Chemical Society 20180108 2

Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be e ...[more]

PMID: 29309130

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Project description:We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding but by hydrophobic and packing forces. These UBPs enabled the creation of the first semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO, we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than, dNaM-dTPT3 in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and especially dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA containing TPT3 and that their improved retention in DNA results in more efficient production of proteins with noncanonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biology "parts" in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and especially dPTMO-dTPT3 represents significant progress toward the development of SSOs with an unrestricted ability to store and retrieve increased information.

Dataset Information

Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

Publications

Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

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