One of the largest challenges in the synthesis of artificial cells that can reproduce is in vitro assembly of ribosomes from in vitro synthesized rRNAs and proteins. In this study, to circumvent the post-transcriptional modification of 16S rRNA for reconstitution of the fully active 30S subunit, we performed artificial evolution of 16S rRNA, which forms the functional 30S subunit without post-transcriptional modifications. We first established an in vitro selection scheme by combining the integrated synthesis, assembly, and translation (iSAT) system with the liposome sorting technique. After 15 rounds of selection cycles, we found one point mutation (U1495C) near the 3′ terminus that significantly enhanced the reconstitution activity of the functional 30S subunit from unmodified 16S rRNA to approximately 57% of that from native-modified 16S rRNA. The effect of the mutation did not depend on the reconstitution scheme, anti-SD sequences, or the target genes to be translated. The mutation we found in this study enabled reconstitution of the active 30S subunit without rRNA modification, and thus would be a useful tool for simple construction of self-reproducing ribosomes.

中文翻译：

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用于简单核糖体重组的未修饰16S rRNA的体外进化

可合成的人工细胞合成中的最大挑战之一是从体外合成的rRNA和蛋白质体外组装核糖体。在这项研究中，为了规避16S rRNA的转录后修饰以重建完全活性的30S亚基，我们进行了16S rRNA的人工进化，形成了没有转录后修饰的功能性30S亚基。我们首先建立了一个体外通过将集成的合成，组装和翻译（iSAT）系统与脂质体分选技术相结合的选择方案。经过15轮选择循环后，我们在3'末端附近发现了一个点突变（U1495C），该突变显着增强了功能性30S亚基从未修饰的16S rRNA的重建活性，至其天然修饰的16S rRNA的重建活性的约57％。突变的效果不取决于重组方案，抗SD序列或待翻译的靶基因。我们在这项研究中发现的突变使得无需rRNA修饰即可重建活跃的30S亚基，因此将成为简单构建自我复制核糖体的有用工具。