Determination of the secondary structure of group II bulge loops using the fluorescent probe 2-aminopurine.

Eleven RNA hairpins containing 2-aminopurine (2-AP) in either base-paired or single nucleotide bulge loop positions were optically melted in 1 M NaCl; and, the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each hairpin were determined. Substitution of 2-AP for an A (adenosine) at a bulge position (where either the 2-AP or A is the bulge) in the stem of a hairpin, does not affect the stability of the hairpin. For group II bulge loops such as AA/U, where there is ambiguity as to which of the A residues is paired with the U, hairpins with 2-AP substituted for either the 5' or 3' position in the hairpin stem have similar stability. Fluorescent melts were performed to monitor the environment of the 2-AP. When the 2-AP was located distal to the hairpin loop on either the 5' or 3' side of the hairpin stem, the change in fluorescent intensity upon heating was indicative of an unpaired nucleotide. A database of phylogenetically determined RNA secondary structures was examined to explore the presence of naturally occurring bulge loops embedded within a hairpin stem. The distribution of bulge loops is discussed and related to the stability of hairpin structures.

Testing the nearest neighbor model for canonical RNA base pairs: revision of GU parameters.

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5'GG/3'UU motif were added to the single representation in the previous database. This revises the ΔG°(37) for the 5'GG/3'UU motif from an unfavorable 0.5 kcal/mol to a favorable -0.2 kcal/mol. Similarly, the ΔG°(37) for the 5'UG/3'GU motif changes from 0.3 to -0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°(37), ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.