Stability of single-nucleotide bulge loops embedded in a GAAA RNA hairpin stem

Forty-six RNA hairpins containing combinations of 3' or 5' bulge loops and a 3' or 5' fluorescein label were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each hairpin were determined. The bulge loops were of the group I variety, in which the identity of the bulge is known, and the group II variety, in which the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The fluorescein label at either the 3' end or 5' end of the hairpin did not significantly influence the stability of the hairpin. As observed with bulge loops inserted into a duplex motif, the insertion of a bulge loop into the stem of a hairpin loop was destabilizing. The model developed to predict the influence of bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability. Specifically, the influence of the bulge is related to the stability of the hairpin stem distal from the hairpin loop.