The relative stabilities of base pair stacking interactions and single mismatches in long RNA measured by temperature gradient gel electrophoresis.

Abstract

The thermal stability of RNA duplexes differing by a single base pair (bp) substitution or mismatch were investigated by temperature gradient gel electrophoresis (TGGE). All base pair substitutions and mismatches were examined at six sites, and limited changes were investigated at three other sites. DNA templates for in vitro transcription were generated by the polymerase chain reaction (PCR). Transcribed forward and reverse single stranded RNAs were annealed to form 345 bp dupex RNA. Solution melting curves of selected RNAs were in good agreement with the predicted three step transitions. Parallel TGGE was used to determine the relative stabilities of the RNAs, and perpendicular TGGE was employed to obtain mobility transitions and midpoint transition temperatures (Tmu) of the RNAs' first melting domain. The gel solvent included formamide and urea. The Tmu values of the first melting domain were influenced by the identity of the base pair substitution or mismatch as well as by the site's neighboring base pairs. The difference in the transition temperatures (deltaTmu) between pairs of RNA ranged from 0 to 5 degrees C. deltaTmu values were used to determine free energy differences (deltaDeltaG). For RNA pairs distinguished by a base pair substitution, the deltaDeltaG values were closely correlated with free energy differences calculated from stacking free energies determined from melting studies in 1 M Na+ [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261.] An algorithm was developed using the free energies of terminal mismatches [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261] that provided very good agreement with experimental free energies for the single internal mismatches.