THE RELATIVE STABILITIES OF BASE-PAIR STACKING INTERACTIONS AND SINGLE MISMATCHES IN LONG RNA MEASURED BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

The thermal stability of RNA duplexes differing by a single base pair (bp) substitution or mismatch were investigated by temperature gradien t gel electrophoresis (TGGE). All base pair substitutions and mismatch es were examined at six sites, and limited changes were investigated a t three other sites. DNA templates for in vitro transcription were gen erated by the polymerase chain reaction (PCR), Transcribed forward and reverse single stranded RNAs were annealed to form 345 bp duper RNA, Solution melting curves of selected RNAs were in good agreement with t he predicted three step transitions. Parallel TGGE was used to determi ne the relative stabilities of the RNAs, and perpendicular TGGE was em ployed to obtain mobility transitions and midpoint transition temperat ures (T-mu) of the RNAs' first melting domain, The gel solvent include d formamide and urea, The T-mu 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 (delta T-mu) between pairs of RNA ranged from 0 to 5 degrees C. delta T-mu values were used to determine free energ y differences (delta Delta G). For RNA pairs distinguished by a base p air substitution, the delta Delta G values were closely correlated wit h free energy differences calculated from stacking free energies deter mined from melting studies in 1 M Na+ [Serra, M. J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261.] An algorithm was developed usi ng the free energies of terminal mismatches [Serra, M, J., and Turner, D. H. (1995) Methods Enzymol. 259, 242-261] that provided very good a greement with experimental free energies for the single internal misma tches.