Thermodynamic characterization of RNA hairpin loop stability

Improved methods for synthesizing oligoribonucleotides have allowed the study of virtually any RNA secondary structure by physical means. RNA secondary structures that have been phylogenetically proven can be used to focus investigations on certain RNA structures. Based on the 16S RNA secondary structure, RNA loops and bulge nucleotides were characterized for their thermodynamic contribution to RNA stability. The study of the stability of phylogenetically proven RNA secondary structures is an important approach to the prediction of RNA structure.; Fourteen RNA hairpin loops containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and a bulge A to the free energy of a hairpin loop is calculated to be 9.3 kcal/mol at 37{dollar}spcirc{dollar}C and succesfully predicts the stability of an independent RNA hairpin loop. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin loop destabilizes an RNA hairpin loop and is sequence dependent. The individual thermodynamic contributions of a four-membered loop and bulge A, G and U residues to the stability of an RNA hairpin loop are presented. A change of G{dollar}cdot{dollar}C to C{dollar}cdot{dollar}G in the loop-closing base pair provides extra stability to the RNA hairpin loop that cannot be explained by nearest-neighbor stacking interactions.; Fifteen RNA hairpin loops varying in loop size from three to nine nucleotides and containing homopolymer loop sequences of A, C and U were synthesized and their thermal stabilities determined. The stability of each loop was deduced by subtracting the known thermodynamic parameters of RNA secondary structure stability from the thermodynamic parameters of hairpin loop formation for each RNA. Tm varys as a function of loop size but is independent of loop composition. Loops of four or five nucleotides are found to be the most stable loop size. This is consistent with the observation that four-membered loops are the most prevalent loop size in 16S-like RNAs.