Sugimoto N, Wu P, Hara H, Kawamoto Y
High Technology Research Center and Department of Chemistry, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada-ku, Kobe 658-8501, Japan.
Biochemistry. 2001 Aug 7;40(31):9396-405. doi: 10.1021/bi010666l.
The effects of cytosine protonation and various cations on the properties of parallel pyrimidine motif DNA triplexes were intensively investigated and characterized by several different techniques, such as circular dichroism (CD) conformation, ultraviolet (UV) melting, differential scanning calorimetry (DSC) thermal denaturation, and surface plasmon resonance (SPR) real-time dynamics. The comparative CD spectra of the triplex and the corresponding homoduplexes showed that the negative peak at approximately 218 nm would be the eigenpeak of the Hoogsteen paired strand, and moreover, the formation pathway of a triplex was significantly pH-dependent and fell into three groups: under acidic conditions, the triplex is formed by a one-step docking, under near physiological conditions, the Watson-Crick duplex is first structured and then accepts the Hoogsteen third strand into its major groove, and under basic conditions, the triplex is not formed. The pH-dependent thermodynamics of the global triplex, the Watson-Crick antiparallel duplex, and the Crick-Hoogsteen parallel duplex were comparatively discussed for the first time. These data revealed that the thermodynamic stabilities of the Watson-Crick-Hoogsteen triplex and the Crick-Hoogsteen duplex would be strongly dependent on cytosine protonation, but a low-pH environment somewhat destabilized the Watson-Crick duplex. The binding energy of triplex formation would be different from the unfolding energy of triplex melting under acidic conditions due to the disparity in the pathway between the formation and unfolding of a triplex. Real-time dynamic measurements showed that the association and dissociation rate constants of a duplex-to-triplex formation are (1.98 +/- 0.24) x 10(3) M(-1) s(-1) and (4.09 +/- 0.96) x 10(-4) s(-1) at 20 degrees C and pH 6.0, respectively. The formation energy of the duplex-to-triplex transition derived from SPR measurements was in agreement with the unfolding energy of the free Hoogsteen paired duplex derived from UV measurements. The calorimetric enthalpies of the triplex-to-duplex-to-single transition were 39.3 and 75.3 kcal/mol under near physiological conditions (pH 7.0), respectively, which were underestimated relative to the van't Hoff enthalpies. In addition, the effects of various cations, ionic strength, mixed-valent cations, and the position of the C(+)xG.C triplets on the thermodynamics of the triplexes were addressed under near physiological conditions. The interaction of metal ions with the triplexes clearly depended on the type and ionic strength of the cations, and the efficiency with which the cations stabilized the global triplex was in the order Mg(2+) > Mn(2+) > Ca(2+) > Ba(2+) >> Na(+). These observations would be useful for the design of triplex-forming oligonucleotides for antigene drugs and therapeutic purposes.
通过圆二色性（CD）构象、紫外（UV）熔解、差示扫描量热法（DSC）热变性和表面等离子体共振（SPR）实时动力学等多种不同技术，对胞嘧啶质子化和各种阳离子对平行嘧啶基序DNA三链体性质的影响进行了深入研究和表征。三链体与相应同源双链体的比较CD光谱表明，约218 nm处的负峰将是Hoogsteen配对链的特征峰，此外，三链体的形成途径显著依赖于pH值，可分为三组：在酸性条件下，三链体通过一步对接形成；在接近生理条件下，沃森-克里克双链体首先形成结构，然后将Hoogsteen第三条链接纳到其大沟中；在碱性条件下，不形成三链体。首次比较讨论了全局三链体、沃森-克里克反平行双链体和克里克-胡斯坦平行双链体的pH依赖性热力学。这些数据表明，沃森-克里克-胡斯坦三链体和克里克-胡斯坦双链体的热力学稳定性将强烈依赖于胞嘧啶质子化，但低pH环境会使沃森-克里克双链体有些不稳定。由于三链体形成和展开途径的差异，酸性条件下三链体形成的结合能与三链体熔解的展开能不同。实时动态测量表明，在20℃和pH 6.0条件下，双链体到三链体形成的缔合和解离速率常数分别为(1.98±0.24)×10³ M⁻¹ s⁻¹和(4.09±0.96)×10⁻⁴ s⁻¹。SPR测量得出的双链体到三链体转变的形成能与UV测量得出的游离Hoogsteen配对双链体的展开能一致。在接近生理条件（pH 7.0）下，三链体到双链体到单链转变的量热焓分别为39.3和75.3 kcal/mol，相对于范特霍夫焓被低估。此外，还研究了接近生理条件下各种阳离子、离子强度、混合价阳离子以及C(+)xG.C三联体位置对三链体热力学的影响。金属离子与三链体的相互作用明显取决于阳离子的类型和离子强度，阳离子稳定全局三链体的效率顺序为Mg²⁺＞Mn²⁺＞Ca²⁺＞Ba²⁺＞＞Na⁺。这些观察结果将有助于设计用于反基因药物和治疗目的的三链体形成寡核苷酸。
