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Transcription of an expanded genetic alphabet.

ABSTRACT: Expansion of the genetic alphabet with a third base pair would have immediate biotechnology applications and also lay the foundation for a semisynthetic organism with an expanded genetic code. A variety of unnatural base pairs have been shown to be formed efficiently and selectively during DNA replication, and the pairs formed between the unnatural nucleotide d5SICS and either dMMO2 or dNaM are particularly interesting because they have been shown to be replicated with efficiencies and fidelities that are beginning to approach those of a natural base pair. Not only are these unnatural base pairs promising for different applications, but they also demonstrate that nucleobase shape and hydrophobicity are sufficient to control replication. While a variety of unnatural base pairs have been shown to be substrates for transcription, none are transcribed in both possible strand contexts, and the transcription of a fully hydrophobic base pair has not been demonstrated. We show here that both of the unnatural base pairs d5SICS:dMMO2 and d5SICS:dNaM are selectively transcribed by T7 RNA polymerase and that the efficiency of d5SICS:dNaM transcription in both possible strand contexts is only marginally reduced relative to that of a natural base pair. Thus, as with replication, we find that hydrogen-bonding is not essential for transcription and may be replaced with packing and hydrophobic forces. The results also demonstrate that d5SICS:dNaM is both replicated and transcribed with efficiencies and fidelities that should be sufficient for use as part of an in vitro expanded genetic alphabet.

SUBMITTER: Seo YJ

PROVIDER: S-EPMC2728119 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Transcription of an expanded genetic alphabet.

Seo Young Jun YJ Matsuda Shigeo S Romesberg Floyd E FE

Journal of the American Chemical Society 20090401 14

Expansion of the genetic alphabet with a third base pair would have immediate biotechnology applications and also lay the foundation for a semisynthetic organism with an expanded genetic code. A variety of unnatural base pairs have been shown to be formed efficiently and selectively during DNA replication, and the pairs formed between the unnatural nucleotide d5SICS and either dMMO2 or dNaM are particularly interesting because they have been shown to be replicated with efficiencies and fidelitie ...[more]

PMID: 19351201

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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.

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Transcription of an expanded genetic alphabet.

Publications

Transcription of an expanded genetic alphabet.

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