In Vitro Evolution of Unmodified 16S rRNA for Simple Ribosome Reconstitution

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.