Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured byUV melting methods. Stability enhancements for rG·dT mismatches were the largest of all mismatchesexamined here, while rU·dG mismatches were not as stable. The methyl group on C5 of thymine enhancedthe stability by 0.12 ~ 0.53 kcal mol
-1 depending on the identity of adjacent Watson-Crick base pairs,whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by ~0.6 kcal mol
-1 regardless of theadjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine,and an nucleotide exchange at rG·dT and rU·dG mismatches were found to be independent of each other.The order for the mismatch stabilities is rG·dT
![](/images/entities/Gt.gif)
rU·dG
![](/images/entities/ap.gif)
rG·dG > rA·dG
![](/images/entities/ap.gif)
rG·dA
![](/images/entities/ap.gif)
rA·dC > rA·dA
![](/images/entities/ap.gif)
rU·dT
![](/images/entities/ap.gif)
rU·dC > rC·dA
![](/images/entities/ap.gif)
rC·dT, although the identity of the adjacent base pairs slightly altered theorder. The pH dependence stability and structural changes were suggested for the rA·dG but not forrG·dA mismatches. Comparisons of trinucleotide stabilities for G·T and G·U pairs in RNA, DNA, andRNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNAmismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches.These results would be useful for the design of antisense oligonucleotides.