Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase,Journal of the American Chemical Society

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Enabling Genetic Code Expansion and Peptide Macrocyclization in mRNA Display via a Promiscuous Orthogonal Aminoacyl-tRNA Synthetase
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-01-11 , DOI: 10.1021/jacs.2c11294
Sabrina E Iskandar _{1,

2} , Jarrett M Pelton _{1,

2} , Elizaveta T Wick _{3,

4} , Derek L Bolhuis _{4,

5} , Albert S Baldwin ₄ , Michael J Emanuele _{3,

4} , Nicholas G Brown _{3,

4} , Albert A Bowers _{1,

2,

6}

Affiliation

mRNA display is revolutionizing peptide drug discovery through its ability to quickly identify potent peptide binders of therapeutic protein targets. Methods to expand the chemical diversity of display libraries are continually needed to increase the likelihood of identifying clinically relevant peptide ligands. Orthogonal aminoacyl-tRNA synthetases (ORSs) have proven utility in cellular genetic code expansion, but are relatively underexplored for in vitro translation (IVT) and mRNA display. Herein, we demonstrate that the promiscuous ORS p-CNF-RS can incorporate noncanonical amino acids at amber codons in IVT, including the novel substrate p-cyanopyridylalanine (p-CNpyrA), to enable a pyridine–thiazoline (pyr–thn) macrocyclization in mRNA display. Pyr–thn-based selections against the deubiquitinase USP15 yielded a potent macrocyclic binder that exhibits good selectivity for USP15 and close homologues over other ubiquitin-specific proteases (USPs). Overall, this work exemplifies how promiscuous ORSs can both expand side chain diversity and provide structural novelty in mRNA display libraries through a heterocycle forming macrocyclization.

中文翻译：

通过混杂正交氨酰基-tRNA 合成酶实现 mRNA 展示中的遗传密码扩展和肽大环化

mRNA 展示通过其快速识别治疗性蛋白质靶标的有效肽结合物的能力，正在彻底改变肽药物的发现。不断需要扩大展示文库化学多样性的方法，以增加鉴定临床相关肽配体的可能性。正交氨酰基-tRNA 合成酶 (ORS) 已被证明可用于细胞遗传密码扩展，但在体外翻译 (IVT) 和 mRNA 展示方面的研究相对较少。在此，我们证明混杂的ORS p -CNF-RS可以在IVT中的琥珀密码子处掺入非规范氨基酸，包括新型底物p -氰基吡啶丙氨酸（ p -CNpyrA），以实现吡啶-噻唑啉（pyr-thn）大环化mRNA 显示。针对去泛素酶 USP15 的基于 Pyr-thn 的选择产生了一种有效的大环结合物，它对 USP15 表现出良好的选择性，并且与其他泛素特异性蛋白酶 (USP) 具有密切的同源性。总体而言，这项工作举例说明了混杂的 ORS 如何通过形成杂环的大环化来扩展侧链多样性并在 mRNA 展示库中提供结构新颖性。

更新日期：2023-01-11

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