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 intron’s 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.