Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.
Expansion of the genetic code through reassignment of redundant sense codons using fully modified tRNA.
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DOI：
10.1093/nar/gkac846
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发表时间：
2022-10-28
影响因子：
14.9
通讯作者：
Hartman, Matthew C. T.
中科院分区：
文献类型：
--
作者：
McFeely, Clinton A. L.;Dods, Kara K.;Patel, Shivam S.;Hartman, Matthew C. T.
为引入非规范氨基酸而打破密码子简并为合成生物学提供了许多机会。然而，尽管存在64个密码子，但该密码子仅在体外扩展到25个氨基酸。一个限制因素可能是过度依赖合成trna，缺乏提高翻译保真度的转录后修饰。为了确定修饰过的野生型tRNA是否能改善意义密码子重配，我们开发了一种新的tRNA捕获方法，并将其应用于大肠杆菌中大约一半的tRNA同工受体的分离。然后，我们在五个捕获的野生型亮基tRNA和它们的合成对应物之间进行密码子竞争实验，揭示了在体外翻译系统中对野生型tRNA的强烈偏好。最后，我们比较了野生型和人工合成的亮氨酸- trna对亮氨酸密码子盒简并性的破坏能力，结果表明只有捕获的野生型trna具有足够的保真度，能够准确地分裂亮氨酸密码子盒，编码三个独立的氨基酸。因此，野生型trna是使基因密码重新分配潜力最大化的试剂。在PURE体外翻译系统中，一种分离修饰的野生型trna的新方法能够有效地将意义密码子重新分配到非规范氨基酸上。
Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for tRNA capture and applied it to the isolation of roughly half of the Escherichia coli tRNA isoacceptors. We then performed codon competition experiments between the five captured wild-type leucyl-tRNAs and their synthetic counterparts, revealing a strong preference for wild-type tRNA in an in vitro translation system. Finally, we compared the ability of wild-type and synthetic leucyl-tRNA to break the degeneracy of the leucine codon box, showing that only captured wild-type tRNAs are discriminated with enough fidelity to accurately split the leucine codon box for the encoding of three separate amino acids. Wild-type tRNAs are therefore enabling reagents for maximizing the reassignment potential of the genetic code. A new method for the isolation of modified, wild-type tRNAs enables efficient reassignment of sense codons to non-canonical amino acids in the PURE in vitro translation system.
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影响因子：
14.9
作者：
Boccaletto P;Stefaniak F;Ray A;Cappannini A;Mukherjee S;Purta E;Kurkowska M;Shirvanizadeh N;Destefanis E;Groza P;Avşar G;Romitelli A;Pir P;Dassi E;Conticello SG;Aguilo F;Bujnicki JM
通讯作者：
Bujnicki JM
影响因子：
64.8
作者：
Fredens, Julius;Wang, Kaihang;Chin, Jason W.
通讯作者：
Chin, Jason W.
DOI：
10.1073/pnas.0509219103
发表时间：
2006-03-21
影响因子：
11.1
作者：
Hartman, MCT;Josephson, K;Szostak, JW
通讯作者：
Szostak, JW
影响因子：
15
作者：
Cui, Zhenling;Stein, Viktor;Alexandrov, Kirill
通讯作者：
Alexandrov, Kirill
影响因子：
2.7
作者：
Kazayama, Ai;Yamagami, Ryota;Hori, Hiroyuki
通讯作者：
Hori, Hiroyuki