LAUSR

Significance Current models for the origin of life include an earlier period when prebiotic chemistry dictated the nonenzymatic copying of RNA polymers, followed by a period of ribozyme-catalyzed reactions using nucleoside-5′-triphosphate (NTP) substrates. Our study addresses the transition from nonenzymatic to ribozyme-catalyzed RNA template copying by determining whether ribozymes could have used substrates activated with 2-aminoimidazole, a prebiotically plausible leaving group, instead of NTPs. We identify ligase ribozymes that use RNA substrates activated with 2-aminoimidazole, thereby proving that ribozyme catalysis is compatible with the reactants of nonenzymatic template-directed ligation. This work suggests that the transition from nonenzymatic to ribozyme-catalyzed RNA replication could have involved ribozymes that utilize inherently reactive RNA substrates. The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in vitro selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide–activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.