Doktycz M J, Paner T M, Amaratunga M, Benight A S
Department of Chemistry, University of Illinois, Chicago 60680.
Biopolymers. 1990;30(7-8):829-45. doi: 10.1002/bip.360300718.
Expressions for the partition function Q (T) of DNA hairpins are presented. Calculations of Q (T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715-1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two-state manner are required. This method is then applied in a systematic manner to investigate the stability of the six basepair duplex stem 5'-GGATAC-3' having four-base dangling single-strand ends with the sequences (XY)2, where X, Y = A, T, G, C, on the 5' end and a T4 loop on the 3' end. Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0 degrees C above the blunt-ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine greater than T greater than C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two-state melting process. Equally accurate fits of all dangling-ended hairpin melting curves were obtained with the two-state model calculation. This was not the case for the melting curve of the blunt-ended hairpin, indicating the presence of a four-base dangling-end drives hairpin melting to a two-state process. Q (T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q (T), the temperature dependence of the transition enthalpy delta H, entropy delta S, and free energy delta G were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879-3885] on the T2 5' dangling-ended d(GC)3 duplexes.(ABSTRACT TRUNCATED AT 400 WORDS)
给出了DNA发夹的配分函数Q(T)的表达式。结合我们之前报道的数值精确算法[T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight(1990)Biopolymers, 29, 1715 - 1734]对Q(T)进行计算，得到了一种直接从DNA发夹的光学熔解曲线评估其转变焓、熵和自由能的温度依赖性的数值方法。无需预先假设短发夹以两态方式熔解。然后以系统的方式应用该方法来研究具有(XY)₂序列的四碱基单链悬垂末端的六碱基对双链体茎5'-GGATAC-3'的稳定性，其中X、Y = A、T、G、C位于5'端，3'端有一个T4环。结果表明，样品集中所有的悬垂末端都能使发夹更稳定，不易熔解。观察到转变温度比平头对照发夹高出多达4.0摄氏度。发夹转变温度的层级顺序由悬垂末端与双链体相邻的第一个碱基的性质决定：嘌呤大于T大于C。通过在数值精确的理论算法中调整单个参数，将每个发夹的计算熔解曲线拟合到实验曲线。在所有情况下都获得了精确的拟合。还假设两态熔解过程计算了实验熔解曲线。两态模型计算对所有悬垂末端发夹的熔解曲线都获得了同样精确的拟合。平头发夹的熔解曲线情况并非如此，这表明四碱基悬垂末端的存在促使发夹熔解为两态过程。使用能使计算曲线与实验获得的光学熔解曲线精确一致的理论参数，计算了每个发夹的Q(T)作为温度的函数。根据Q(T)，计算了每个发夹的转变焓ΔH、熵ΔS和自由能ΔG的温度依赖性，从而对悬垂末端对发夹热力学的影响进行了定量评估。将我们的结果与Breslauer小组[M. Senior, R. A. Jones, and K. J. Breslauer(1988)Biochemistry 27, 3879 - 3885]关于T2 5'悬垂末端d(GC)₃双链体的结果进行了比较。(摘要截断于400字)
