Blaskó A, Minyat E E, Dempcy R O, Bruice T C
Department of Chemistry, University of California, Santa Barbara 93106, USA.
Biochemistry. 1997 Jun 24;36(25):7821-31. doi: 10.1021/bi970064v.
Short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2) and the guanidinium (g) linked thymidyl nucleoside d(Tg)4-T-azido associate as triplexes. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters and activation energies for the triplexes were determined by UV thermal analysis. The hypochromic shift and Tm for triplex formation increases with increase in concentration and decreases with the number of mismatches. The melting temperatures are between 35 and 55 degrees C in the range of ionic strength of 0.06-0.24 and decrease with increase in ionic strength at 100 deg/(ionic strength unit). The melting and cooling curves exhibit hysteresis behavior in the temperature range 5-95 degrees C at 0.2 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (k(on), E(on)) and dissociation (k(off), E(off)) processes were obtained. The second-order rate constants, k(on), for the triplex formation at 288 K are between 10 and 500 M(-2) s(-1). Values of k(on) increase with the decrease in the ionic strength. The first order rate constants for the dissociation, k(off), at 288 K are between 10(-6) and 40 x 10(-6) s(-1) and increase with increase in ionic strength. The energies of activation for the association and dissociation processes are in the range -22 to -9 kcal/mol and 8 to 29 kcal/mol, respectively. At 6.3 x 10(-5) M/base and at the physiological ionic strength (0.15-0.30) and below, the triplex structures formed with d(Tg)4-T-azido and A5G3A5 and GA4G3A4G have well-defined Tm values. The melting curves with G2A3G3A3G2 and G2A2G5A2G2 are very shallow with small hypochromic shifts denoting negligible binding at physiological ionic strength. Therefore, with the increase in the G content (mismatched base pairs) at a certain concentration (e.g., 6.3 x 10(-5) M/base), discrimination (change in fidelity) occurs in the formation and strength of binding of d(Tg)4-T-azido to d(pAn pGm) oligomers. The standard molar enthalpies for triplex formation have in general larger negative values at low ionic strength than at high ionic strength, indicating that at lower mu values the formation of triplexes of d(Tg)4-T-azido with d(pAn pGm) are more favorable. The values of deltaH(standard)(288) calculated from the activation parameters are between -17 and -49 kcal/mol, and the values of deltaG(standard)(288) are between -7.5 and -11.8 kcal/mol for A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2, respectively. There is a linear relationship in the enthalpy-entropy compensation for the triplex melting thermodynamics.
短链DNA寡聚物（A5G3A5、GA4G3A4G、G2A3G3A3G2和G2A2G5A2G2）与胍基（g）连接的胸腺嘧啶核苷d（Tg）4 - T - 叠氮化物形成三链体。通过紫外热分析测定了三链体的解链温度（Tm）、缔合和解离动力学及热力学参数以及活化能。三链体形成时的减色效应和Tm值随浓度增加而增大，随错配数增加而减小。在离子强度为0.06 - 0.24范围内，解链温度在35至55摄氏度之间，且在离子强度每增加100度/（离子强度单位）时随离子强度增加而降低。在热速率为0.2度/分钟的情况下，在5至95摄氏度温度范围内，熔解曲线和冷却曲线呈现滞后行为。从这些曲线中，获得了缔合（k（on），E（on））和解离（k（off），E（off））过程的速率常数和活化能。在288 K时，三链体形成的二级速率常数k（on）在10至500 M⁻² s⁻¹之间。k（on）值随离子强度降低而增大。在288 K时，解离的一级速率常数k（off）在10⁻⁶至40×10⁻⁶ s⁻¹之间，且随离子强度增加而增大。缔合和解离过程的活化能分别在 - 22至 - 9 kcal/mol和8至29 kcal/mol范围内。在6.3×10⁻⁵ M/碱基以及生理离子强度（0.15 - 0.30）及以下时，由d（Tg）4 - T - 叠氮化物与A5G3A5和GA4G3A4G形成的三链体结构具有明确界定的Tm值。与G2A3G3A3G2和G2A2G5A2G2形成的熔解曲线非常平缓，减色效应小，表明在生理离子强度下结合可忽略不计。因此，在一定浓度（例如6.3×10⁻⁵ M/碱基）下，随着G含量（错配碱基对）增加，d（Tg）4 - T - 叠氮化物与d（pAn pGm）寡聚物结合的形成和强度会出现区分（保真度变化）。三链体形成的标准摩尔焓在低离子强度下通常比在高离子强度下具有更大的负值，表明在较低的μ值下，d（Tg）4 - T - 叠氮化物与d（pAn pGm）形成三链体更有利。根据活化参数计算得到的288 K时的ΔH（标准）值在 - 17至 - 49 kcal/mol之间，对于A5G3A5、GA4G3A4G、G2A3G3A3G2和G2A2G5A2G2，ΔG（标准）（288）值分别在 - 7.5至 - 11.8 kcal/mol之间。三链体熔解热力学的焓 - 熵补偿存在线性关系。
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