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The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°(37), ΔH° and melting temperature (T(m)) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG &gt; rU·dT &gt; rA·dA &gt; rC·dC. The stability trend for the base pairs 5' of the single internal mismatch is rG·dC &gt; rC·dG &gt; rA·dT &gt; rU·dA. The stability trend for the base pairs 3' of the single internal mismatch is rC·dG &gt; rG·dC &gt;&gt; rA·dT &gt; rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes