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.