Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217-3364, USA.
Int J Mol Sci. 2021 Jan 18;22(2):895. doi: 10.3390/ijms22020895.
Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to codons in Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved machinery are compared with related measurements employing the orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the tyrosyl and pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the system will highlight the most promising positions at which the orthogonal pair may infiltrate the genetic code.
遗传密码扩展主要集中在重新分配琥珀终止密码子，以将非标准氨基酸（ncAAs）的单拷贝插入蛋白质中。越来越多的努力被用于利用先前进化为琥珀酸抑制的氨酰 tRNA 合成酶（aaRS）变体，以响应密码子在 DH10B 中以 98.1±3.6%的效率重新编程琥珀终止密码子，与野生型酪氨酸掺入的正交对的效率相媲美。新的正交对被部署用于快速评估我们之前开发的荧光基础筛选中的有义密码子重排潜力。使用进化后的机器测量有义密码子重排效率，并与使用相关的正交对系统进行比较。重要的是，我们观察到酪氨酸的和吡咯赖氨酸的系统有不同的有义密码子重排效率模式，这表明特定的密码子将更适合不同的正交对进行重排。使用 系统对酪氨酸进行广泛的有义密码子重排效率评估将突出正交对可能渗透到遗传密码的最有前途的位置。
