A semisynthetic organism engineered for the stable expansion of the genetic alphabet
A semisynthetic organism engineered for the stable expansion of the genetic alphabet
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DOI：
10.1073/pnas.1616443114
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发表时间：
2017-02-07
影响因子：
11.1
通讯作者：
Romesberg, Floyd E.
中科院分区：
文献类型：
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作者：
Zhang, Yorke;Lamb, Brian M.;Romesberg, Floyd E.
所有天然生物体都以由4种字母、2种碱基对组成的遗传字母表来存储遗传信息。通过添加2种能选择性配对形成非天然碱基对（UBP）的合成非天然核苷酸来扩充遗传字母表，将提升DNA的信息存储潜力，而稳定携带这种扩充字母表的半合成生物体（SSO）将因此具备存储和检索更多信息的潜力。为实现这一目标，我们此前报道过，在非天然核苷三磷酸dNaMTP和d5SICSTP存在的条件下培养大肠杆菌，并通过表达质粒携带的核苷三磷酸转运蛋白为其提供摄取这些物质的途径，大肠杆菌能够复制含有单个dNaM - d5SICS UBP的DNA。尽管这是一项重要的概念验证，但这种新生的SSO生长状况不佳，更麻烦的是，它需要在可控条件下生长，即便如此也无法无限期存储非天然信息，而这显然是真正的半合成生命的一个先决条件。在此，为强化并赋予这种新生的SSO活力，我们对转运蛋白进行了改造，采用了化学性质更优化的UBP，并利用细菌免疫反应的力量，通过Cas9去除丢失了UBP的DNA。优化后的SSO生长旺盛，能持续摄取非天然三磷酸核苷，并且几乎能在任何序列环境中无限期保留多个UBP。因此，这种SSO是一种能够利用由6种字母、3种碱基对组成的字母表稳定存储遗传信息的生命形式。
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.