Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro.
Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro.
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DOI：
10.1093/nar/gky365
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发表时间：
2018-07-06
影响因子：
14.9
通讯作者：
Alexandrov K
中科院分区：
文献类型：
--
作者：
Cui Z;Wu Y;Mureev S;Alexandrov K
正义密码子重分配为非天然氨基酸（uAA）代表了用于将新性质引入多肽的有力方法。这种方法的主要障碍是天然同种受体tRNA和正交tRNA之间对重新分配的密码子的竞争。虽然存在几种用于在无细胞翻译系统中选择性失活tRNA异源受体的色谱和酶促方法，但它们复杂且不可扩展。我们设计了一组由脱氧、核糖或2′-O-甲基核糖核苷酸组成的tRNA反义寡核苷酸，并测试了它们有效复合所选tRNA的能力。靶向反密码子和tRNASerGCU可变环之间序列的甲基化寡核苷酸显示出亚纳摩尔的结合亲和力和缓慢的解离动力学。这样的寡核苷酸有效地和选择性地螯合天然tRNASerGCU直接在有免疫活性的大肠杆菌S30裂解物中，从而消除其翻译活性并释放AGU/AGC密码子。在tRNASerGCU缺失的E.大肠杆菌裂解液允许其用n-炔丙基-l-赖氨酸或p-叠氮基-l-苯丙氨酸进行同质修饰。本文所开发的策略是通用的，如在E. coli翻译系统。此外，该方法具有种间无关性，并成功应用于真核动物利什曼原虫的体外翻译系统，为遗传密码重分配研究提供了一个新的方向，具有广泛的实际应用价值。
Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2′-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylalanine. The strategy developed here is generic, as demonstrated by sequestration of tRNAArgCCU isoacceptor in E. coli translation system. Furthermore, this method is likely to be species-independent and was successfully applied to the eukaryotic Leishmania tarentolae in vitro translation system. This approach represents a new direction in genetic code reassignment with numerous practical applications.
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影响因子：
14.9
作者：
Chan PP;Lowe TM
通讯作者：
Lowe TM
影响因子：
15
作者：
Cui, Zhenling;Stein, Viktor;Alexandrov, Kirill
通讯作者：
Alexandrov, Kirill
影响因子：
3.2
作者：
Ahn, Jin-Ho;Hwang, Mi-Yeon;Kim, Dong-Myung
通讯作者：
Kim, Dong-Myung
影响因子：
56.9
作者：
ECKER, DJ;VICKERS, TA;ZOUNES, M
通讯作者：
ZOUNES, M
DOI：
10.1006/bbrc.2000.2556
发表时间：
2000-04-21
影响因子：
3.1
作者：
Kanda, T;Takai, K;Takaku, H
通讯作者：
Takaku, H