Stability Studies on Random Folded RNAs (‘Never Born RNAs’), Implications for the RNA World

In the last years, we have developed a method for the study of random RNA sequences with the aim of investigating their capacity to assume folded and possibly functionally active (ribozymes) structures. The RNA Foster assay developed in our laboratory is a powerful, simple, and fast method to investigate the structural properties of RNA by assessing the presence of secondary domains and their thermal stability through the combination of S1 nuclease activity at different temperatures. In this work, we investigated the structural properties of totally de novo random RNAs, 97‐nucleotides‐long, of which 60 nucleotides were completely random. The rationale behind this was to assess whether and to what extent random RNAs would acquire a stable fold as a prerequisite for catalytic activity. A library of random sequences was created, and 18 sequences were randomly selected and analyzed. Surprisingly, most of the RNAs were resistant to S1 nuclease digestion at up to 50°, and two sequences were resistant even at 70°, suggesting that these totally random RNA molecules could posses a stable secondary structure over a broad range of temperatures under the conditions used. We discuss the possible relevance of these data for the general scenario of the RNA world.