Nature2014-05-21 5:27 PM

 具有扩展遗传字母的半合成生物 A semi-synthetic organism with an expanded genetic alphabet

论文摘要


生物受它们基因组中编码的信息所定义,并且自生命起源,该信息通过使用两对碱基对遗传字母(A-T和G-C)而被编码。在体外,遗传字母已扩大到了包含多个人工碱基对(UBPS)。以d5SICS和DNAM(d5SICS-DNAM)之间形成的碱基对为例,我们已经开发出一类在承载疏水性核碱基的核苷酸之间形成的UBP,这能在体外进行有效PCR扩增和体外转录,且其独特的复制机制已经得到表征。然而,生物体遗传字母的扩展提出了前所未有的新挑战:非自然核苷三磷酸必须可在细胞内获得;内源性聚合酶必须能够在复杂细胞环境中使用该非自然核苷三磷酸来忠实地复制包含UBP的DNA ;最后UBP在维持DNA完整性的通路中必须是稳定的。在这里我们表明,外生性表达的藻类核苷三磷酸转运子能够有效将d5SICS和DNAM(d5SICSTP和dNaMTP)中的三磷酸导入大肠杆菌,并且内源性复制机制使用它们来准确地复制含有d5SICS-DNAM的质粒。非自然三磷酸的存在和UBP的复制都无法导入显著的增长负担。最后我们发现,UBP不能通过DNA修复途径有效地被切除。因此,所得细菌是首个能稳定传播扩展遗传字母的生物。


Abstract


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.


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