Crowdsourcing RNA Re-Design Enables Discovery of Ribosomes with Beneficial Phenotypes Under Diverse Environmental Conditions

The ribosome polymerizes amino acids into proteins in a sequence-defined and highly accurate manner. Its capabilities could be harnessed for the sustainable synthesis of any type of polymer. However, understanding and engineering the ribosome has been challenging - at its heart are three RNAs (rRNAs), facilitating mRNA decoding and amino acid polymerization. The recently developed cell-free platform iSAT permits rapid iteration on ribosome design-build-test-learn, accelerating ribosome engineering attempts. Still, the gigantic rRNA sequence space combined with a poor understanding of the relationship of rRNA sequence, folding, and function limits the success of conventional methods for designing functional rRNAs. For an alternative approach to designing functional rRNAs, we turn to Eterna, an online video game that crowdsources RNA sequence design to citizen scientists in the form of puzzles based on secondary structure energetics. Together, we transformed the traditional design-build-test-learn approach. By applying our new approach, we discovered 16S and 23S rRNA sequences that improve ribosome performance under diverse environmental conditions in vitro and in vivo. These novel ribosomes can provide new insights into the ribosome’s rRNA sequence-folding-function relationship, be exploited for the sustainable production of next generation therapeutics and materials, and endow cells with beneficial environmental phenotypes.