Stability of 3′ double nucleotide overhangs that model the 3′ ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3′ overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3′ terminal overhang relative to the single 3′ terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37°C of the second base in the double 3′ terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3′ double overhang can contribute nearly the same stability to a duplex as a base pair or a 3′ dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3′ end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3′ purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3′ double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.