Singleton S F, Dervan P B
Arnold and Mabel Beckman Laboratories of Chemical Synthesis, California Institute of Technology, Pasadena 91125.
Biochemistry. 1992 Nov 17;31(45):10995-1003. doi: 10.1021/bi00160a008.
The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.
在22摄氏度（100 mM Na⁺和1 mM精胺）、pH值范围为5.8至7.6的条件下，采用定量亲和切割滴定法测定了嘧啶·嘌呤·嘧啶结构基序的寡聚脱氧核糖核苷酸导向三链螺旋形成的能量学。5'-TTTTTCTCTCTCTCT-3'（1）和5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3'（2，m5C为2'-脱氧-5-甲基胞苷）的平衡结合常数在pH从7.6降至5.8时分别增加了10倍和20倍，这表明相应的三链螺旋复合物在较低pH值下分别稳定了1.4和1.7 kcal·mol⁻¹。用5-甲基胞嘧啶残基取代1中的五个胞嘧啶残基得到2，在pH值5.8至7.6范围内，三链螺旋稳定了0.1至0.4 kcal·mol⁻¹。根据一般pH依赖性平衡转变的定量模型对这些数据进行分析表明，含有胞苷和5-甲基胞苷的嘧啶寡核苷酸分别形成局部三链螺旋结构，其表观pKa值分别为5.5（C+GC三联体）和5.7（m5C+GC三联体），并且这些寡核苷酸即使在中性pH以上也应以约10⁶ M⁻¹的表观亲和常数与大DNA上的单个位点结合。
