Moe J G, Russu I M
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459.
Biochemistry. 1992 Sep 15;31(36):8421-8. doi: 10.1021/bi00151a005.
Proton nuclear magnetic resonance (NMR) spectroscopy is used to characterize the kinetics and energetics of base-pair opening in the dodecamers 5'-d(CGCGAATTCGCG)-3' and 5'-d(CGCGAATTTGCG)-3'. The latter dodecamer contains two symmetrical G.T mismatched base pairs. The exchange kinetics of imino protons is measured from resonance line widths and selective longitudinal relaxation times. For the G.T pair, the two imino protons (G-N1H and T-N3H) provide probes for the opening of each base in the mismatched pair. The lifetimes of individual base pairs in the closed state and the equilibrium constants for formation of the open state are obtained from the dependence of the exchange rates on the concentration of ammonia catalyst. The activation energies and standard enthalpy changes for base-pair opening are obtained from the temperature dependence of the lifetimes and equilibrium constants, respectively. The results indicate that the G.T mismatched pairs are kinetically and energetically destabilized relative to normal, Watson-Crick base pairs. The lifetimes of the G.T pairs are of the order of 1 ms or less, over the temperature range from 0 to 20 degrees C. The equilibrium constants for base-pair opening, at 20 degrees C, are increased up to 4000-fold, relative to those of normal base pairs. The energetic destabilization of the G.T base pairs is, at least in part, enthalpic in origin. The presence of the G.T mismatched base pairs destabilizes also neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)
质子核磁共振（NMR）光谱法用于表征十二聚体5'-d（CGCGAATTCGCG）-3'和5'-d（CGCGAATTTGCG）-3'中碱基对打开的动力学和能量学。后一个十二聚体包含两个对称的G.T错配碱基对。亚氨基质子的交换动力学通过共振线宽和选择性纵向弛豫时间来测量。对于G.T碱基对，两个亚氨基质子（G-N1H和T-N3H）为错配碱基对中每个碱基的打开提供了探针。从交换速率对氨催化剂浓度的依赖性中获得单个碱基对在闭合状态下的寿命以及打开状态形成的平衡常数。碱基对打开的活化能和标准焓变分别从寿命和平衡常数的温度依赖性中获得。结果表明，相对于正常的沃森-克里克碱基对，G.T错配碱基对在动力学和能量学上是不稳定的。在0至20摄氏度的温度范围内，G.T碱基对的寿命约为1毫秒或更短。在20摄氏度时，碱基对打开的平衡常数相对于正常碱基对增加了多达4000倍。G.T碱基对的能量不稳定至少部分源于焓。G.T错配碱基对的存在也使相邻碱基对不稳定。（摘要截断于250字）
