RNA i-Motif Formation at Neutral pH

i-Motifs are non-canonical nucleic acid secondary structures formed in sequences rich in cytosine. Previous work has shown that DNA i-motifs may form at neutral pH and provided evidence to suggest they influence biological functions. RNA i-motifs are less stable than DNA i-motifs, so more questions surround the biological relevance of these structures in RNA. Using biophysical methods, we found that increasing cytosine tract lengths resulted in increased thermal stability but not pH stability. In ensemble solution experiments RNA i-motifs appear globally unfolded at neutral pH. However, single molecule experiments revealed that 1% of RNA i-motifs remain folded in solution. This has implications for the potential of formation of RNA i-motifs in cells.