Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA.
Nat Biotechnol. 2020 Aug;38(8):989-999. doi: 10.1038/s41587-020-0479-2. Epub 2020 Apr 13.
A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of noncanonical biopolymers.
在扩展细胞的遗传密码以将非规范氨基酸掺入蛋白质中时，一个核心挑战是可扩展地发现氨酰-tRNA 合成酶（aaRS）-tRNA 对，它们在氨酰化特异性方面是正交的。在这里，我们通过计算从数百万个序列中识别候选正交 tRNA，并开发了一种快速、可扩展的方法——tRNA 扩展（tREX）——来确定 tRNA 的体内氨酰化状态。使用 tREX，我们在大肠杆菌中测试了 243 个候选 tRNA，并鉴定了 71 个正交 tRNA，涵盖了 16 个同工受体类别和 23 个功能正交 tRNA-aaRS 对。我们发现了五个正交对，包括三个高度活跃的琥珀色抑制物，并为两对进化了新的氨基酸底物特异性。最后，我们使用 tREX 来描述 64 个正交合成酶-正交 tRNA 特异性的矩阵。这项工作扩展了可用于遗传密码扩展的正交对的数量，并为发现其他正交对提供了一个途径，并为细胞中非规范生物聚合物的合成提供了基础。
