The ability to site-specifically incorporate two distinct noncanonical amino acids (ncAAs) into the proteome of a mammalian cell with high fidelity and efficiency will have many enabling applications. It would require the use of two different engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs, each suppressing a distinct nonsense codon, and which cross-react neither with each other, nor with their counterparts from the host cell. Three different aaRS/tRNA pairs have been developed so far to expand the genetic code of mammalian cells, which can be potentially combined in three unique ways to drive site-specific incorporation of two distinct ncAAs. To explore the suitability of using these combinations for suppressing two distinct nonsense codons with high fidelity and efficiency, here we systematically investigate: (1) how efficiently the three available aaRS/tRNA pairs suppress the three different nonsense codons, (2) preexisting cross-reactivities among these pairs that would compromise their simultaneous use, and (3) whether different nonsense-suppressor tRNAs exhibit unwanted suppression of non-cognate stop codons in mammalian cells. From these comprehensive analyses, two unique combinations of aaRS/tRNA pairs emerged as being suitable for high-fidelity dual nonsense suppression. We developed expression systems to validate the use of both combinations for the site-specific incorporation of two different ncAAs into proteins expressed in mammalian cells. Our work lays the foundation for developing powerful applications of dual-ncAA incorporation technology in mammalian cells, and highlights aspects of this nascent technology that need to be addressed to realize its full potential.

中文翻译：

---

定义哺乳动物细胞中双重非经典氨基酸诱变的当前范围和局限性

以高保真度和高效率将两个不同的非经典氨基酸（ncAAs）特异地掺入哺乳动物细胞的蛋白质组中的能力将具有许多有利的应用。这将需要使用两个不同的工程改造的氨酰基-tRNA合成酶（aaRS）/ tRNA对，每个对都抑制一个独特的无义密码子，并且它们彼此之间或与宿主细胞中的对应物都不会发生交叉反应。迄今为止，已经开发了三种不同的aaRS / tRNA对来扩展哺乳动物细胞的遗传密码，可以潜在地以三种独特的方式进行组合，以驱动两个不同的ncAA的位点特异性结合。为了探索使用这些组合以高保真度和效率抑制两个不同的无义密码子的适用性，在这里我们系统地研究：（1）三个可用的aaRS / tRNA对抑制三个不同的无义密码子的效率如何；（2）这些对之间预先存在的交叉反应性会损害它们的同时使用；以及（3）不同的无义抑制性tRNA是否表现出不希望的抑制作用哺乳动物细胞中的非同源终止密码子。从这些综合分析中，出现了两种独特的aaRS / tRNA对组合，适用于高保真双重无义抑制。我们开发了表达系统，以验证将两种不同的ncAAs特异性结合到哺乳动物细胞表达的蛋白质中，两种组合的使用。我们的工作为开发双重NCAA整合技术在哺乳动物细胞中的强大应用奠定了基础，