Ribosome-dependent protein biosynthesis is an essential cellular process mediated by transfer RNAs (tRNAs). Generally, ribosomally synthesized proteins are limited to the 22 proteinogenic amino acids (pAAs: 20 l-α-amino acids present in the standard genetic code, selenocysteine, and pyrrolysine). However, engineering tRNAs for the ribosomal incorporation of non-proteinogenic monomers (npMs) as building blocks has led to the creation of unique polypeptides with broad applications in cellular biology, material science, spectroscopy, and pharmaceuticals. Ribosomal polymerization of these engineered polypeptides presents a variety of challenges for biochemists, as translation efficiency and fidelity is often insufficient when employing npMs. In this Review, we will focus on the methodologies for engineering tRNAs to overcome these issues and explore recent advances both in vitro and in vivo. These efforts include increasing orthogonality, recruiting essential translation factors, and creation of expanded genetic codes. After our review on the biochemical optimizations of tRNAs, we provide examples of their use in genetic code manipulation, with a focus on the in vitro discovery of bioactive macrocyclic peptides containing npMs. Finally, an analysis of the current state of tRNA engineering is presented, along with existing challenges and future perspectives for the field.

中文翻译：

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用于非蛋白原单体核糖体翻译的工程 tRNA


核糖体依赖性蛋白质生物合成是由转移 RNA (tRNA) 介导的重要细胞过程。一般来说，核糖体合成的蛋白质仅限于 22 个蛋白氨基酸（pAA：标准遗传密码中存在的 20 个 l-α-氨基酸、硒代半胱氨酸和吡咯赖氨酸）。然而，对 tRNA 进行改造，将非蛋白原单体 (npM) 作为构件掺入核糖体中，已经产生了独特的多肽，在细胞生物学、材料科学、光谱学和制药领域具有广泛的应用。这些工程多肽的核糖体聚合给生物化学家带来了各种挑战，因为在使用 npM 时翻译效率和保真度往往不足。在这篇综述中，我们将重点关注工程 tRNA 的方法，以克服这些问题，并探索体外和体内的最新进展。这些努力包括增加正交性、招募必要的翻译因子以及创建扩展的遗传密码。在对 tRNA 的生化优化进行回顾之后，我们提供了它们在遗传密码操作中的应用示例，重点是含有 npM 的生物活性大环肽的体外发现。最后，分析了 tRNA 工程的现状，以及该领域现有的挑战和未来的前景。