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A semi-synthetic organism with an expanded genetic alphabet

机译：具有扩展的遗传字母的半合成生物

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摘要

遗传代码很简单: 形成两对的四个碱基(A-T和G-C)被用在所有生命中。对这一代码加以扩展、将非天然核苷酸和碱基对也包括在内一直是合成生物学的一个目标，因为它将为出于特定目的定制生物铺平道路。虽然这一点已在体外原理证明实验中做到，但一个扩展的代码的稳定传播此前一直没有在活体中演示过。Floyd Romesberg及同事提供的证据表明，两种憎水核苷酸d5SICSTP和dNaMTP能够被添加到表达一种外源藻类三磷酸核苷运输分子的大肠杆菌在其内生长的介质中，这些核苷酸将会被吸收到基因组中，而不会被修复通道当成病变。因此，含有DNA的非天然碱基对被复制，而细胞生长不会受到显著影响。%Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
机译：遗传代码很简单: 形成两对的四个碱基(A-T和G-C)被用在所有生命中。对这一代码加以扩展、将非天然核苷酸和碱基对也包括在内一直是合成生物学的一个目标，因为它将为出于特定目的定制生物铺平道路。虽然这一点已在体外原理证明实验中做到，但一个扩展的代码的稳定传播此前一直没有在活体中演示过。 Floyd Romesberg及同事提供的证据表明，两种憎水核苷酸d5SICSTP和dNaMTP能够被添加到表达一种外源藻类三磷酸核苷运输分子的大肠杆菌在其内生长的介质中，这些核苷酸将会被吸收到基因组中，而不会被修复通道当成病变。因此，含有DNA的非天然碱基对被复制，而细胞生长不会受到显着影响。 %Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (AT and GC). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we sho w that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.

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  • 来源
    《Nature》 |2014年第7500期|385-388a2|共5页
  • 作者

    Denis A. Malyshev; Kirandeep Dhami; Thomas Lavergne; Tingjian Chen; Nan Dai; Jeremy M. Foster; Ivan R. Correa Jr; Floyd E. Romesberg;

  • 作者单位

    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;

    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;

    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;

    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;

    New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA;

    New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA;

    New England Biolabs, 240 County Road, Ipswich, Massachusetts 01938, USA;

    Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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