Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nucleic Acids Res. 2019 May 21;47(9):e54. doi: 10.1093/nar/gkz143.
Structural analysis of ribosomes in complex with aminoacyl- and/or peptidyl-transfer RNA (tRNA) often suffers from rapid hydrolysis of the ester bond of aminoacyl-tRNAs. To avoid this issue, several methods to introduce an unhydrolyzable amide bond instead of the canonical ester bond have been developed to date. However, the existing methodologies require rather complex steps of synthesis and are often inapplicable to a variety of amino acids including those with noncanonical structures. Here, we report a new method to synthesize 3'-aminoacyl-NH-tRNAs by means of flexizymes-ribozymes capable of charging amino acids onto tRNAs. We show that two types of flexizymes, dFx and eFx, are able to charge various amino acids, including nonproteinogenic ones, onto tRNA or microhelix RNA bearing the 3'-deoxy-3'-amino-adenosine. Due to the versatility of the flexizymes toward any pair of nonproteinogenic amino acids and full-length or fragment tRNAs, this method provides researchers an opportunity to use a wide array of hydrolytically stable 3'-aminoacyl-NH-tRNAs and analogs for various studies.
核糖体与氨酰基-和/或肽基-tRNA（tRNA）复合物的结构分析常常受到氨酰基-tRNA 酯键快速水解的影响。为了避免这个问题，目前已经开发了几种引入不可水解的酰胺键而不是经典酯键的方法。然而，现有的方法学需要相当复杂的合成步骤，并且通常不适用于包括具有非经典结构的氨基酸在内的各种氨基酸。在这里，我们报告了一种通过能够将氨基酸加载到 tRNA 上的 flexizymes-ribozymes 合成 3'-氨酰基-NH-tRNA 的新方法。我们表明，两种类型的 flexizymes，dFx 和 eFx，能够将各种氨基酸，包括非蛋白氨基酸，加载到 tRNA 或带有 3'-脱氧-3'-氨基-腺苷的 microhelix RNA 上。由于 flexizymes 对任何一对非蛋白氨基酸和全长或片段 tRNA 的多功能性，这种方法为研究人员提供了使用广泛的水解稳定的 3'-氨酰基-NH-tRNA 和类似物进行各种研究的机会。
