Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl–transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl–tRNA synthetase (PylRS)/^PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/^ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ^ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ^ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/^ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/^PyltRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.

扩展和重新编程细胞的遗传密码以整合多个不同的非规范氨基酸（ncAAs），以及编码的非规范生物聚合物的生物合成，需要发现多个正交的氨酰基转移RNA合成酶/ tRNA对。这些对必须与宿主合成酶和tRNA相互正交。Pyrrolysyl–tRNA合成酶（PylRS）/ ^Pyl tRNA对是遗传密码扩展最广泛使用的系统。在这里，我们揭示了ΔNPylRS/ ^ΔNPyltRNA对的序列（缺少N末端域）形成了两个不同的类别。我们显示，测得的ΔNPylRSs和^{ΔNPyltRNA的}特异性与基于序列的聚类相关，并且大多数ΔNPylRSs优先与所属类别的^ΔNPyltRNA。然后，我们从88个ΔNPylRS/ ^ΔNPyltRNA组合中鉴定出18个相互正交的对。此外，我们生成了一组12个三对正交对，每个对由三个新的PylRS / ^Pyl tRNA对组成。最后，我们在三重正交集内使每对的ncAA特异性和解码特性不同，并指导将三个不同的非经典氨基酸整合到单个多肽中。