Enzymatic aminoacylation of tRNA with unnatural amino acids
Enzymatic aminoacylation of tRNA with unnatural amino acids
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DOI：
10.1073/pnas.0509219103
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发表时间：
2006-03-21
影响因子：
11.1
通讯作者：
Szostak, JW
中科院分区：
文献类型：
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作者：
Hartman, MCT;Josephson, K;Szostak, JW
细胞翻译装置的生物化学灵活性原则上为合成类药物修饰多肽和新型生物聚合物提供了一条简单的途径。然而，只有大约75个非自然的构建基团与酶促tRNA酰化和随后的核糖体修饰肽的合成完全相容。尽管翻译系统可以在合成多肽的过程中的几个步骤中拒绝底物类似物，但大部分特异性存在于氨基酰-tRNA合成酶(AARS)的水平上，该酶负责向tRNA充电氨基酸。我们已经开发了一种基于质谱学的AARS分析方法，可以用来快速鉴定可以被酶荷到tRNA上的非天然单体。通过使用这种方法，我们已经找到了59种未知的AARS底物。其中包括许多具有有用功能性质的侧链类似物。值得注意的是，许多β-氨基酸、N-甲基氨基酸以及α，α-二取代氨基酸也是AARS的底物。这些以前未鉴定的AARS底物将有助于研究后续翻译步骤的特异性，并可能显著增加可用于修饰肽的核糖体合成的类似物的数量。
The biochemical flexibility of the cellular translation apparatus offers, in principle, a simple route to the synthesis of drug-like modified peptides and novel biopolymers. However, only approximate to 75 unnatural building blocks are known to be fully compatible with enzymatic tRNA acylation and subsequent ribosomal synthesis of modified pepticles. Although the translation system can reject substrate analogs at several steps along the pathway to peptide synthesis, much of the specificity resides at the level of the aminoacyl-tRNA synthetase (AARS) enzymes that are responsible for charging tRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly identify unnatural monomers that can be enzymatically charged onto tRNA. By using this assay, we have found 59 previously unknown AARS substrates. These include numerous side-chain analogs with useful functional properties. Remarkably, many beta-amino acids, N-methyl amino acids, and alpha,alpha-disubstituted amino acids are also AARS substrates. These previously unidentified AARS substrates will be useful in studies of the specificity of subsequent steps in translation and may significantly expand the number of analogs that can be used for the ribosomal synthesis of modified pepticles.