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bioRxiv · 10.1101/2025.05.06.652512

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

Biswas, A.·Weiss, Z.·Szostak, J. W.·DasGupta, S.
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Abstract

Ribozymes played essential roles in catalyzing metabolic processes and facilitating genome replication in primordial RNA-based life. In vitro evolution has enabled the discovery of ribozymes that catalyze diverse chemical reactions, but it is likely that many catalytic activities of RNA remain to be uncovered. Expanding our understanding of the biochemical capabilities of RNA, especially new ribozyme functions related to RNA assembly, will help to refine models of primordial genome replication and, more broadly, the origin and evolution of early life. Here, we report the discovery of a novel ribozyme ligase that catalyzes the attack of the 2'-hydroxyl group of an RNA substrate on the 5'-triphosphate of a second RNA, but only when the substrate RNA possesses a 3'-phosphate vicinal to its nucleophilic 2'-hydroxyl group. This activity emerged unexpectedly during a directed evolution experiment designed to isolate ribozyme ligases that use 5'-triphosphorylated RNA oligonucleotides as substrates. Because the 3'-phosphate group on the RNA substrate does not directly participate in the ligation reaction, it would be extremely challenging to design a selection strategy to isolate ribozyme ligases with this unique reactivity. The ligases requirement for an RNA 3'-phosphate group on the substrate resembles enzymatic mechanisms found in protein-based RNA repair pathways. We propose that ribozyme-catalyzed ligation of 3'-phosphorylated RNA could have enabled the repair of cleaved RNA strands in primordial cells. We demonstrate that our in vitro evolved ribozymes ligate themselves specifically to 3'-phosphorylated RNA fragments present in heterogenous mixtures of cellular RNA, demonstrating their potential utility as enrichment reagents for profiling RNA cleavage products in transcriptomic 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.

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Biswas, A., Weiss, Z., Szostak, J. W., DasGupta, S.. 2025-05-08. A ribozyme ligase that requires a 3' terminal phosphate on its RNA substrate. https://doi.org/10.1101/2025.05.06.652512

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