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Trinucleotide bulges in RNA commonly occur in nature. Yet, little data exists concerning the thermodynamic parameters of this motif. Algorithms that predict RNA secondary structure from sequence currently attribute a constant free energy value of 3.2 kcal/mol to all trinucleotide bulges, regardless of bulge sequence. To test the accuracy of this model, RNA duplexes that contain frequent naturally occurring trinucleotide bulges were optically melted, and their thermodynamic parameters-enthalpy, entropy, free energy, and melting temperature-were determined. The thermodynamic data were used to derive a new model to predict the free energy contribution of trinucleotide bulges to RNA duplex stability: ΔG°37, trint bulge = ΔG°37, bulge + ΔG°37, AU + ΔG°37, GU. The parameter ΔG°37, bulge is variable depending upon the purine and pyrimidine composition of the bulge, ΔG°37, AU is a 0.49 kcal/mol penalty for an A-U closing pair, and ΔG° 37, GU is a -0.56 kcal/mol bonus for a G-U closing pair. With both closing pair and bulge sequence taken into account, this new model predicts free energy values within 0.30 kcal/mol of the experimental value. The new model can be used by algorithms that predict RNA free energies as well as algorithms that use free energy minimization to predict RNA secondary structure from sequence.

Improved Model to Predict the Free Energy Contribution of Trinucleotide Bulges to RNA Duplex Stability