Esplorazione funzionale e strutturale dello spazio di sequenze dell'RNA. : implicazioni per l'origine della vita e le biotecnologie

The RNA World hypothesis, which assumes that an RNA World preceded our contemporary DNA/RNA/protein World, has become more and more popular in the field of the origin of life (Joyce, 2002; Orgel, 2003). Despite the recent progresses made in this field, some basic questions remain unanswered: Can the RNA catalyze the reactions needed for self-replication on the early Earth? Can the RNA-based life achieve the metabolic sophistication needed to give birth to the protein-nucleic acid World? To tackle these questions a number of theoretical and experimental works (Szostack et al., 2001; Muller, 2006) have been carried out with the ultimate goal to re-create an RNA World in laboratory. Within this framework lies the “Never Born Biopolymers (NBBs)” project (Luisi et al., 2006) and in particular the “Never Born RNAs” (NBRs), project which has the goal to explore the RNA sequence space for catalytic functions. This project moves from the observation that the extant collection of RNA molecules is only a minor part of the all theoretical possible RNA sequences (Luisi, 2003). On the basis of this observation, the question whether a functionality is a common feature or a rare result of natural selection is of the utmost importance to elucidate the role of RNA in the origin of Life and to fully exploit its biological potential. Knowing the firm relation between structure and function of biological molecules we decided to precede the RNAs functional exploration with some structural studies using the RNA Foster (RNA Folding Stability Test) (De Lucrezia et al., 2006a). This assay employs S1 nuclease, a specific nuclease (Vogt, 1973) to cleave at different temperatures single-strand RNA sequences, monitoring the presence of double-stranded domains and indirectly any possible structure. In fact, folded RNAs are more resistant to S1 nuclease than unfolded ones, namely the latter are degraded faster than the former. In addition, we exploited the capability of nuclease S1 to work over a broad range of temperatures to probe RNA secondary domain stability at different conditions. In fact, an increase in temperature destabilizes the RNA fold, inducing either global or local unfolding. Consequently, the RNA becomes susceptible to nuclease attack and is readily degraded. In few words the most stable sequences at high temperature will be those with a more stable secondary and possibly tertiary structure. 3 Until now the most general result of our studies lies in the demonstration that RNAs have the capacity to fold into compact secondary structures, even in absence of selective pressure (Anella et al., 2011). This confirm our hypothesis that molecules involved in nowadays life don’t have exclusive features at far as the ability to adopt a stable fold is concerned. In detail one of the sequence analyzed has a stability higher than the tRNA at 70°C , with an approximately melting temperature higher than 80°C. The results will be used to provide directions and suggestions for further studies concerning the functional properties of RNAs in the early evolution scenario