In vitro selection of RNAs with increased tertiary structure stability

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Abstract
An in vitro selection system was devised to select RNAs based on their tertiary structural stability, independent of RNA activity. Selection studies were conducted on the P4–P6 domain from the Tetrahymena thermophila group I intron, an autonomous self-folding unit that contains several important tertiary folding motifs including the tetraloop receptor and the A-rich bulge. Partially randomized P4–P6 molecules were selected based on their ability to fold into compact structures using native gel electrophoresis in the presence of decreasing concentrations of MgCl2. After 10 rounds of the selection process, a number of sequence alterations were identified that stabilized the P4–P6 RNA. One of these, a single base deletion of C209 within the P4 helix, significantly stabilized the P4–P6 molecule and would not have been identified by an activity-based selection because of its essential role for ribozyme function. Additionally, the sequence analysis provided evidence that stabilization of secondary structure may contribute to overall tertiary stability for RNAs. This system for probing RNA structure irrespective of RNA activity allows analysis of RNA structure/function relationships by identifying nucleotides or motifs important for folding and then comparing them with RNA sequences required for function.