In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid
In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid
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DOI：
10.1021/ja048200o
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发表时间：
2004-12-15
影响因子：
15
通讯作者：
Sisido, M
中科院分区：
文献类型：
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作者：
Ninomiya, K;Minohata, T;Sisido, M
用非天然氨基酸实现tRNA特异性化学氨酰化。一种非天然氨基酸被激活为硫酯衍生物，后者通过间隔区连接到9-mer肽核酸的N-末端，所述9-mer肽核酸与酵母苯丙氨酸tRNA的X-末端区域互补。当氨基酸硫酯-间隔基-PNA缀合物与tRNA混合时，观察到有效的氨酰化。PNA辅助的氨酰化也成功地在大肠杆菌在体外蛋白质合成系统，其中包含一个正交的tRNA。原位氨酰化/体外翻译得到了一个突变蛋白，其中非天然氨基酸被纳入到由CGGG 4-碱基coclon/anticoclon对指导的位置。
tRNA-specific chemical aminoacylation was achieved with nonnatural amino acids. A nonnatural amino acid was activated as a thioester derivative, and the latter was linked through a spacer to the N-terminal of a 9-mer peptide nucleic acid that is complementary to the X-terminal region of yeast phenylalanine tRNA. Efficient aminoacylation was observed when the amino acid thioester-spacer-PNA conjugate was mixed with the tRNA. The PNA-assisted aminoacylation was also successful in an Escherichia coli in vitro protein synthesizing system that contained an orthogonalized tRNA. The in situ aminoacylation/ in vitro translation gave a mutant protein in which the nonnatural amino acid was incorporated into the position directed by a CGGG 4-base coclon/anticoclon pair.