PDF(Pubmed)
Abstract：
Ribosomal incorporation of β-amino acids into nascent peptides is much less efficient than that of the canonical α-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved β-amino acid incorporation. However, multiple/consecutive incorporations of β-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of β-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of β-homophenylglycine (βPhg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive βPhg\'s. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for β-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of β-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple β-amino acids.
摘要：
β-氨基酸在新生肽中的核糖体掺入效率远低于经典α-氨基酸。为了克服这一点，我们设计了一个带有tRNAGlu的T-茎和tRNAPro1的D-臂的tRNA嵌合体，称为tRNAPro1E2，它有效地募集EF-Tu和EF-P。使用tRNAPro1E2确实改善了β-氨基酸掺入。然而,β-氨基酸的多次/连续掺入仍然不利地较差。这里,我们尝试对tRNAPro1E2的反密码子臂进行微调，旨在进一步增强β-氨基酸的掺入。通过筛选引入tRNAPro1E2的各种突变，C31G39/C28G42突变显示CCG密码子处连续两次掺入β-高苯基甘氨酸（βPhg）的大约3倍增强。这种tRNA的使用使得第一次有可能延长多达十个连续的βPhg。由于反密码子臂突变的增强效果因用于β-氨基酸掺入的密码子而异，我们优化了五个密码子的反密码子臂序列(CCG，CAU,CAG,ACU和UGG）。这些密码子的五种最佳tRNA的组合使得有可能将五种不同种类的β-氨基酸和类似物同时引入模型肽中。包括大环支架。该策略将使含有多个β-氨基酸的大环肽文库的核糖体合成成为可能。
