Szokoli, D. | Search bioRxiv

bioRxiv2026

Cryo-EM of a nucleotide-polymerizing ribozyme enables its predictive improvement

Ribozymes capable of self-replication from nucleotides would have been central to the hypothesized RNA World. The leading laboratory models for such molecules were converted from a class I ligase by in vitro evolution but then developed without 3D structures. Here, scaffolded cryo-EM of the substrate-free tC19Z RNA polymerase ribozyme at 3.1 [A] resolution shows how this conversion was achieved. An accessory domain evolved from random sequence grips the ancestral ligase through a loop-loop contact, a seam of magnesium ions, and a six-base stack, and rebuilds the ligases substrate binding site from different residues of its own. A previously unrecognized pairing, present before substrate binds, sequesters the 5' end that must otherwise pair with the template. Compensatory mutations to the ribozyme and template, designed to break this ectopic pairing, increase the extension rate. These results suggest that accelerating RNA structure determination may speed progress toward nucleotide-based self-replication.

biochemistry↗

bioRxiv2024

Protein-Free Catalysis of DNA Hydrolysis and Self-Integration by a Ribozyme

Group II introns are ancient self-splicing ribozymes and retrotransposons. Though long speculated to have originated before translation, their dependence on intron-encoded proteins for splicing and mobility has cast doubt on this hypothesis. While some group II introns are known to retain part of their catalytic repertoire in the absence of protein cofactors, protein-free complete reverse splicing of a group II intron into a DNA target has never been demonstrated. Here, we demonstrate the complete independence of a group II intron from protein cofactors in all intron-catalyzed reactions. The ribozyme is capable of fully reverse splicing into single-stranded DNA targets in vitro, readily hydrolyzes DNA substrates, and is even able to unwind and react with stably duplexed DNA. Our findings make a protein-free origin for group II introns plausible by expanding their known catalytic capabilities beyond what would be needed to survive the transition from RNA to DNA genomes. Furthermore, the introns capacity to react with both single and double-stranded DNA in conjunction with its expanded sequence recognition may represent a promising starting point for the development of protein-free genomic editing tools.

biochemistry↗