category
NAR
date
Feb 24, 2026
slug
status
Published
summary
通过工程改造tRNA的反密码子茎，显著提高非天然氨基酸（如N-甲基氨基酸和d-氨基酸）的核糖体掺入效率；筛选出11种高效突变体，可同时提升多种非天然氨基酸的掺入能力；成功实现含连续三组非天然氨基酸的环肽合成，为构建多组分异源氨基酸肽库提供工具。
tags
基因编辑
合成生物学
type
Post
📄 原文题目
Engineering of tRNAPro1E2 anticodon stem enhances multiple/consecutive ribosomal incorporation of N-methyl-l-α-amino acids and d-α-amino acids
🔗 原文链接
💡 AI 核心解读
通过工程改造tRNA的反密码子茎，显著提高非天然氨基酸（如N-甲基氨基酸和d-氨基酸）的核糖体掺入效率；筛选出11种高效突变体，可同时提升多种非天然氨基酸的掺入能力；成功实现含连续三组非天然氨基酸的环肽合成，为构建多组分异源氨基酸肽库提供工具。
📝 英文原版摘要
<span class="paragraphSection"><div class="boxTitle">Abstract</div>Transfer RNA (tRNA) structures influence the incorporation efficiency of amino acids, particularly when nonproteinogenic amino acids (npAAs), such as <span style="font-style: italic;">N</span>-methyl-<span style="font-variant: small-caps;">l</span>-α-amino acids (<sup>Me</sup>AAs) and <span style="font-variant: small-caps;">d</span>-α-amino acids (<span style="font-variant: small-caps;">d</span>-AAs), are charged. Such npAAs are generally far poorer substrates than the <span style="font-variant: small-caps;">20</span> canonical α-amino acids for translation. However, we have shown that their incorporation efficiencies could be improved by using a chimeric tRNA, termed tRNA<sup>Pro1E2</sup>, bearing optimal T-stem and D-arm. Here we report that the engineering anticodon stem of tRNA<sup>Pro1E2</sup> further enhances multiple/consecutive elongation of <sup>Me</sup>AAs and <span style="font-variant: small-caps;">d</span>-AAs. By screening 149 types of anticodon stem mutants, we found eleven tRNA<sup>Pro1E2</sup> variants capable of enhancing the incorporation of <span style="font-style: italic;">N</span>-methyl-<span style="font-variant: small-caps;">l</span>-leucine (<sup>Me</sup>Leu) or <span style="font-variant: small-caps;">d</span>-phenylalanine (<span style="font-variant: small-caps;">d</span>-Phe) at six codons by up to 4.5- and 4.4-fold, respectively. Interestingly, 7 out of the 11 variants showed different mobilities compared to the parental tRNAs<sup>Pro1E2</sup> in native polyacrylamide gel electrophoresis analysis, indicating that their unique conformations contributed to promoting peptidyl transfer reaction. These tRNAs exhibited increased incorporation efficiencies of not only <sup>Me</sup>Leu
and <span style="font-variant: small-caps;">d</span>-Phe but also diverse <sup>Me</sup>AAs and <span style="font-variant: small-caps;">d</span>-AAs. Ribosomal synthesis of a model macrocyclic peptide containing three consecutive <sup>Me</sup>AAs and three <span style="font-variant: small-caps;">d</span>-AAs was also demonstrated with high quality, indicating their usefulness for the peptide library construction containing multiple exotic amino acids.</span>
- 作者:NotionNext
- 链接:https://tangly1024.com/article/31348bd6-1f96-811a-98b2-ee11265df854
- 声明:本文采用 CC BY-NC-SA 4.0 许可协议，转载请注明出处。
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