Evolutionary optimization of a modular ligase ribozyme: a small catalytic unit and a hairpin motif masking an element that could form an inactive structure

The YFL ribozyme is an artificial ligase ribozyme isolated by a ‘design and selection’ strategy, in which a modular catalytic unit was generated on a rationally designed modular scaffold RNA. This ligase ribozyme has a versatile catalytic unit that accepts not only β-nicotinamide mononucleotide (β-NMN) but also inorganic pyrophosphate as leaving groups for template-dependent RNA ligation. Although this property is interesting from an evolutionary viewpoint regarding primitive RNA ligation/polymerization systems in the RNA world, structural analysis of the YFL ribozyme has not been continued due to apparent structural nonuniformity of its folded state. To elucidate the active structure of the YFL ribozyme, we performed in vitro evolution experiments to improve its folding ability. Biochemical and phylogenetic analyses of evolved variants indicated that the catalytic unit of the YFL ribozyme is compact and the 3′ single-stranded region of the parent YFL-1 ribozyme contributes to mask an element that could form an inactive structure.