Department of Life Science, College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan.
Int J Mol Sci. 2020 Oct 21;21(20):7793. doi: 10.3390/ijms21207793.
Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in . A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.
遗传密码系统的重编程受到构建新 tRNA 结构的困难限制。在这里，我利用报告基因检测系统，开发了带有小发夹 RNA 适体标签的翻译活性 tRNA 变体。作为工程用的 tRNA 底盘，我采用了具有 12 碱基对氨基酸接受臂的 9/3 组成和长可变臂（V 臂）的 allo-tRNA 的琥珀酸抑制变体。尽管它们的 V 臂是丝氨酸 tRNA 合成酶（SerRS）的强结合位点，但在 V 臂茎区插入一个突环核苷酸会阻止 allo-tRNA 分子被 SerRS 用丝氨酸进行负载。SerRS 拒绝的 allo-tRNA 底盘被设计成具有丙氨酸、酪氨酸或组氨酸的另一种氨基酸身份。V 臂的尖端被替换为不同的发夹 RNA 适体，这些适体被表达在 中的同源蛋白识别。高亲和力相互作用导致 allo-tRNA 分子被隔离，而中等亲和力的适体部分则招募与同源蛋白结构域融合的组氨酰-tRNA 合成酶变体。这种用于 tRNA-适体融合的新设计原则将增强遗传密码的激进和动态操作。
