The effect of single base-pair mismatches on the duplex stability of d(T-A-T-T-A-A-T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A).
The stabilitye and dynamics of the duplex d(T-A-T-T-A-A--T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A) has been studied by means of ultraviolet-melting, temperature-jump relaxation kinetics, stopped-flow and NMR spectroscopy. In addition, the influence of the mismatches A . A, G . T, A .C and T . C,-incorporated in this double helix through the introduction of non-complementary bases in the second strand, on these parameters has been investigated. The thermodynamic parameters characterizing the melting of the duplexes have been determined. Interestingly, a substantial decrease was observed for the values of the melting enthalpy when proceeding from 0.015 M to 0.1 M NaCl solutions. All duplexes that contain mismatches have melting temperatures below that of the totally complementary double helix. On the basis of NMR experiments and differences in the free enthalpy values between the totally complementary double helix and the duplexes with mismatches, it could be concluded that some degree of stacking of the two mispaired bases between the neighbouring base pairs is maintained. At 1 M NaCl the enthalpy and entropy of the helix-to-coil transition of the totally complementary double helix are in good agreement with values calculated on the basis of the thermodynamic data of Borer et al. [Borer, Ph. N., Dengler, B. & Tinoco, I. (1974) J. Mol. Biol. 86, 843--853] which were derived for RNA. The kinetics of the complementary duplex and duplexes with G . T and A . C mismatches were studied by means of stopped-flow and temperature-jump techniques. The rate constants of formation are the same for the three double helices. The decrease in stability of the duplexes with mismatches is therefore entirely due to an increase of the dissociation constant. In temperature-jump experiments very often a fast relaxation process is observed in addition to the relaxation characterizing the disruption of the double helix. This fast relaxation process is customarily attributed to base destacking in the single helix. By combining temperature-jump relaxation kinetics with NMR melting experiments, it is shown that at the low temperature side of the melting transition this fast relaxation process is caused by rapid changes in the double-helical structure.
D001482:Base Composition; D001483:Base Sequence; D005815:Genetic Code; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D009841:Oligonucleotides