A ribozyme ligase that requires a 3′ terminal phosphate on its RNA substrate

Abstract

Abstract Ribozymes likely played essential roles in catalyzing metabolic processes and facilitating genome replication in primordial RNA-based life. In vitro evolution has allowed us to expand the biochemical capabilities of RNA, especially new ribozyme chemistries. Here, we report the serendipitous discovery of ribozyme ligases that catalyze the attack of the 2′-hydroxyl group of an RNA substrate on its own 5′-triphosphate group, but only when the substrate possesses a 3′-phosphate vicinal to its nucleophilic 2′-hydroxyl group. The ligases′ requirement for a 3′-phosphate group on its substrate resembles enzymatic mechanisms found in protein-based RNA repair pathways. We propose that ribozyme-catalyzed ligation of 3′-phosphorylated RNA could have provided pathways for RNA repair in primordial cells. We demonstrate that these ribozymes ligate specifically to 3′-phosphorylated RNA present in a heterogeneous mixture of cellular RNAs. We further show that these ribozymes can capture cleaved RNAs with 3′-phosphate and 2′−3′-cyclic phosphate termini, enabling us to selectively amplify the captured RNAs. These results demonstrate their potential utility as enrichment reagents for profiling RNA cleavage products in transcriptomics studies. Our findings not only report a new catalytic reactivity in RNA but also provide insights into ribozyme evolution, primordial RNA repair, and potential applications in RNA sequencing.