Goto Yuki, Suga Hiroaki
Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2012;848:465-78. doi: 10.1007/978-1-61779-545-9_29.
Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to non-proteinogenic ones, and thus specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. We here describe a protocol that facilitates the genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of non-proteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-acyl groups, D: -amino acids, N-methyl amino acids, and physiologically stable macrocyclic scaffolds. Facility of the protocol allows for a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions.
遗传密码重编程是一种将任意密码子从蛋白质氨基酸重新分配为非蛋白质氨基酸的方法，因此可以根据其mRNA模板核糖体表达特定序列的非标准肽。我们在此描述一种方案，该方案使用与定制的体外翻译装置集成的柔性酶促进遗传密码重编程，该装置称为柔性体外翻译（FIT）系统。柔性酶是灵活的tRNA酰化核酶，能够制备各种非蛋白质酰基-tRNA。这些酰基-tRNA读取FIT系统中产生的空缺密码子，产生具有各种奇异结构的所需非标准肽，例如N-酰基、D-氨基酸、N-甲基氨基酸和生理稳定的大环支架。该方案的便利性允许在具有生物学功能的新型非标准肽的合成中进行广泛的应用。
