Use of 2‐Aminopurine to detect positional ambiguity in group II single‐ nucleotide bulge loops

Abstract

Six RNA hairpin sequences containing a single‐nucleotide bulge loop with the fluorescent analog 2‐aminopurine (2‐AP) were optically melted in 1M NaCl and subject to fluorescent spectroscopy. Four of the bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity in the exact position of the bulge. The structural similarity to adenine and the fluorescent nature and properties of 2‐AP were used to analyze the uncertainty in position by inserting the fluorophore into the bulge loop on the 5′ and 3′ sides of the hairpin stem. The two remaining hairpins act as controls, with one containing a group I bulge loop and the other being fully paired. The group I bulge loop, which contains an unpaired nucleotide, showed a decrease in fluorescence as temperature increased, where the fully paired hairpin showed an increase in fluorescence with temperature. Previously, structural probing of group II bulge loops within hairpin motifs (McCann et al. 2011) indicated that the bulged nucleotide is the one positioned farther from the hairpin loop. This study suggests that when 2‐AP exists in group II bulge loops as the nucleotide closer to the hairpin loop, it exhibits behavior indicative of an unpaired nucleotide.