Thermodynamics of coaxially stacked helixes with GA and CC mismatches.

The thermodynamics of RNA helix-helix interfaces with intervening single- and tandem-GA or single-CC mismatches were studied by UV melting experiments. The model system consists of a hairpin with a four- or five-nucleotide 5' overhang which is bound by a short oligomer, creating the helical interface. Single GA interfaces are found to have favorable free energy increments of about 2 kcal/mol. This is similar to those reported for coaxially stacked flush interfaces of AU base pairs [Walter A. E., & Turner, D. H. (1994) Biochemistry 33, 12715-12719]. The free energy increment of the GA mismatches depends little on the sequence of the closing base pairs of the helixes, whether the break in the phosphate backbone is 5' or 3' with respect to the mismatch or whether the chains are extended beyond the helix-helix interface. Surprisingly, interfaces with single-CC mismatches have favorable free energy increments similar to those of GA interfaces, even though CC mismatches in coaxial stacks occur much less frequently in known RNA secondary structures. The results provide experimental support for the assumption that a bonus free energy is required for coaxially stacked helixes with intervening GA mismatches when free energy minimization is used to predict RNA secondary structures.