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A semisynthetic organism engineered for the stable expansion of the genetic alphabet.

ABSTRACT: All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5307467 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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A semisynthetic organism engineered for the stable expansion of the genetic alphabet.

Zhang Yorke Y Lamb Brian M BM Feldman Aaron W AW Zhou Anne Xiaozhou AX Lavergne Thomas T Li Lingjun L Romesberg Floyd E FE

Proceedings of the National Academy of Sciences of the United States of America 20170123 6

All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escher ...[more]

PMID: 28115716

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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

A semisynthetic organism engineered for the stable expansion of the genetic alphabet.

Publications

A semisynthetic organism engineered for the stable expansion of the genetic alphabet.

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