Computational De Novo Design of Group II Introns Yields Highly Active Ribozymes

Group II introns (G2Is) are large self-splicing ribozymes with emerging potential in biotechnological applications. Despite growing interest, their complexity has so far precluded efforts to design them from scratch. While computational approaches have enabled the design of small RNA catalysts, methods for engineering large ribozymes remain underdeveloped. Herein, the RNA inverse folding algorithm aRNAque is used to design G2Is from scratch, yielding three novel self-splicing ribozymes with unusually stable structures. The designed intron Arq.I2 is revealed to be an unexpectedly proficient ribozyme in vitro, self-splicing at a rate comparable to the fastest known G2Is. While most G2Is are believed to be inactive under intracellular conditions in the absence of maturase proteins, it is shown that Arq.I2 self-splices in Escherichia coli cells. The results demonstrate that highly active variants of large and complex ribozymes can be designed de novo with relative ease using existing inverse folding algorithms, paving the way for the design of bespoke ribozymes derived from G2Is for the development of biotechnological tools.