The hammerhead self-cleaving motif as a precursor to complex endonucleolytic ribozymes.

Connections between distinct catalytic RNA motifs through networks of mutations that retain catalytic function (neutral networks) were likely central to the evolution of biocatalysis. Despite suggestions that functional RNAs collectively form an interconnected web of neutral networks, little evidence has emerged to demonstrate the existence of such intersecting networks in naturally occurring RNAs. Here we show that neutral networks of two naturally occurring, seemingly unrelated endonucleolytic ribozymes, the hammerhead (HH) and hairpin (HP), intersect. Sequences at the intersection of these networks exhibit catalytic functions corresponding to both ribozymes by potentially populating both catalytic folds and enable a smooth crossover between the two. Small and structurally simple endonucleolytic motifs like the HH ribozyme could, through mutational walks along their neutral networks, encounter novel catalytic phenotypes, and structurally flexible, bifunctional sequences at the intersection of these networks could have acted as nodes for evolutionary diversification in an RNA world. Considering the simplicity and small size of the HH ribozyme, we propose that this self-cleaving motif could have been a precursor to other more complex endonucleolytic ribozymes. More generally, our results suggest that RNAs that possess distinct sequences, structures, and catalytic functions, can potentially share evolutionary history through mutational connections in sequence space.