THE THERMAL-STABILITY OF DNA FRAGMENTS WITH TANDEM MISMATCHES AT A D(CXYG)CENTER-DOT-D(CY'X'G) SITE

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determ ine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson-Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacen t mismatches were formed by melting and reannealing pairs of homologou s 373 bp DNA fragments differing by two adjacent base pairs. Product D NAs were separated based on their thermal stability by parallel and pe rpendicular TGGE, The polyacrylamide gel contained 3.36 M urea and 19. 2% formamide to lower the DNA melting temperatures. The order of stabi lity was determined in the sequence context d(CXYG). d(CY'X'G) where X . X' and Y . Y' represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions i nfluence DNA stability, Mobility transition melting temperatures (T-u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 d egrees C) lower than the homoduplex DNA with the d(CCAG). d(CTGG) sequ ence. Two adjacent G . A pairs, d(CGAG). d(CGAG), created a more stabl e DNA than DNAs with Watson-Crick A . T pairs at the same sites. The d (GA). d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more sta ble in free energy than d(AA). d(TT) base pairs. This result confirms the unusual stability of the d(GA). d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative sta bilities followed an order expected from previous results on single mi smatches. Two homoduplex DNAs with identical nearest neighbor sequence s but different next-nearest neighbor sequences had a small but reprod ucible difference in T-u value. This result indicates that sequence de pendent next neighbor stacking interactions influence DNA stability.