Quadruplex-Flanking Stem Structures Modulate the Stability and Metal Ion Preferences of RNA Mimics of GFP

The spinach family of RNA aptamers
are RNA mimics of green fluorescent
protein (GFP) that have previously been designed to address the challenges
of imaging RNA inside living cells. However, relatively low levels
of free intracellular magnesium limited the practical use of these
aptamers. Recent cell-based selections identified the broccoli RNA
aptamer, which requires less magnesium for fluorescence, but the basis
for magnesium preference remained unclear. Here, we find that the
broccoli RNA structure is very similar to that of baby spinach, a
truncated version of the spinach aptamer. Differences in stability
and metal ion preferences between these two aptamers, and among broccoli
mutants, are primarily associated with the sequence and structure
of predicted quadruplex-flanking stem structures. Mutation of purine–purine
pairs in broccoli at the terminal stem–quadruplex transition
caused reversion of broccoli to a higher magnesium dependence. Unique
duplex-to-quadruplex transitions in GFP-mimic RNAs likely explain
their sensitivity to magnesium for stability and fluorescence. Thus,
optimizations designed to improve aptamers should take into consideration
the role of metal ions in stabilizing the transitions and interactions
between independently folding RNA structural motifs.