THE CONTRIBUTION OF DNA SINGLE-STRANDED ORDER TO THE THERMODYNAMICS OF DUPLEX FORMATION
THE CONTRIBUTION OF DNA SINGLE-STRANDED ORDER TO THE THERMODYNAMICS OF DUPLEX FORMATION
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DOI：
10.1073/pnas.88.9.3569
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发表时间：
1991-05-01
影响因子：
11.1
通讯作者：
BRESLAUER, KJ
中科院分区：
文献类型：
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作者：
VESNAVER, G;BRESLAUER, KJ
我们报告了DNA单链顺序对DNA双链形成的热力学贡献的直接测定。通过差示扫描量热法（DSC）和温度相关的紫外吸收光谱，我们对13聚双相[d(CGCATGAGTACGC)]的热诱导破坏进行了热力学表征。[d(GCGTACTCATGCG)]（以下称为S1）。S2)及其组成单链[d(CGCATGAGTACGC)]（以下称为S1）和[d(GCGTACTCATGCG)]（以下称为S2）。这些光谱和量热测量得到了以下25℃时的热力学曲线：δ g度= 20.0 kcal/mol， δ h度= 117.0 kcal/mol， δ s度= 325.4 cal.度-1 mol-1；δ - g度= 0.45 kcal/mol， δ - h度= 29.1 kcal/mol， δ -s度= 96.1 cal.degree-1 mol-1；单链熔融S2 (1 cal = 4.184 J) δ g度= 1.44 kcal/mol， δ h度= 27.2 kcal/mol， δ s度= 86.4 cal.degree-1 mol-1。这些数据表明，尽管表现出相当大的转变焓，但两个单链结构S1和S2仅在25℃时稳定。这种行为是由于焓和熵的贡献大小相似，它们相互补偿，从而导致单链在25℃时稳定的自由能相对较小。相比之下，S1。S2双相态在25℃时非常稳定，因为与双相断裂相关的有利转变熵（325.4 cal.degree-1 mol-1）被极大的双相转变焓（117.0 kcal/mol）所补偿。我们还使用等温批量混合量热法直接测量了在25℃下双相生成的焓变（δ h度）为-56.4 kcal/mol。在25℃时双相生成焓为-56.4 kcal/mol，与在74℃时用DSC测定的双相破坏焓为117.0 kcal/mol有很大的不同。由于DSC测量显示净转变热容变化接近于零，我们将双链破坏和双链形成焓之间的巨大差异解释为反映了25℃（等温混合实验中的初始状态）单链结构和几乎等于80℃（DSC实验中的最终状态）单链结构的差异。事实上，25℃下双链形成的焓（-56.4 kcal/mol）可以与从25℃到80℃融化每条单链所需的积分焓之和（S1为23.6 kcal/mol， S2为27.2 kcal/mol）相结合，计算出假设的25℃下“随机线圈”“非堆叠”单链双链形成过程的delta - h度为-107.2 kcal/mol。该预测的双相形成的delta - h度值的大小与我们通过DSC直接测量的双相破坏的相应参数（117.0 kcal/mol）非常吻合，从而为我们对数据的解释和分析提供了可信度。因此，我们的研究结果表明，尽管单链在25℃下仅具有边际稳定性，但单链可以表现出分子内相互作用，从而使它们在焓上稳定地形成双链。对于本文研究的双链，在25℃下结合之前，两条互补的单链已经拥有了总焓（50.8/117）的40%，最终稳定了最终的双链状态。单链结构在室温附近的这一特征可以显著降低人们可能从最近邻数据中预测双链形成的焓驱动力，因为这些数据通常来自单链处于随机线圈状态的测量。因此，在设计杂交实验和使用等温滴定和/或批量混合技术研究双链和高阶DNA结构（如三链、四链等）的形成时，必须认识和考虑单链结构的潜在贡献。
We report a direct determination of the thermodynamic contribution that DNA single-stranded order makes to DNA duplex formation. By using differential scanning calorimetry (DSC) and temperature-dependent UV absorbance spectroscopy, we have characterized thermodynamically the thermally induced disruption of the 13-mer duplex [d(CGCATGAGTACGC)].[d(GCGTACTCATGCG)] (henceforth called S1.S2) and its component single strands, [d(CGCATGAGTACGC)] (henceforth called S1) and [d(GCGTACTCATGCG)] (henceforth called S2). These spectroscopic and calorimetric measurements yield the following thermodynamic profiles at 25-degrees-C: DELTA-G-degrees = 20.0 kcal/mol, DELTA-H-degrees = 117.0 kcal/mol, and DELTA-S-degrees = 325.4 cal.degree-1.mol-1 for duplex melting of S1.S2; DELTA-G-degrees = 0.45 kcal/mol, DELTA-H-degrees = 29.1 kcal/mol, and DELTA-S-degrees = 96.1 cal.degree-1.mol-1 for single-strand melting of S1; DELTA-G-degrees = 1.44 kcal/mol, DELTA-H-degrees = 27.2 kcal/mol, and DELTA-S-degrees = 86.4 cal.degree-1.mol-1 for single-strand melting of S2 (1 cal = 4.184 J). These data reveal that the two single-stranded structures S1 and S2 are only marginally stable at 25-degrees-C, despite exhibiting rather substantial transition enthalpies. This behavior results from enthalpy and entropy contributions of similar magnitudes that compensate each other, thereby giving rise to relatively small free energies of stabilization for the single strands at 25-degrees-C. By contrast, the S1.S2 duplex state is very stable at 25-degrees-C since the favorable transition entropy associated with duplex disruption (325.4 cal.degree-1.mol-1) is more than compensated for by the extremely large duplex transition enthalpy (117.0 kcal/mol). We also measured directly an enthalpy change (DELTA-H-degrees) of -56.4 kcal/mol for duplex formation at 25-degrees-C using isothermal batch-mixing calorimetry. This duplex formation enthalpy of -56.4 kcal/mol at 25-degrees-C is very different in magnitude from the duplex disruption enthalpy of 117.0 kcal/mol measured at 74-degrees-C by DSC. Since the DSC measurement reveals the net transition heat capacity change to be close to zero, we interpret this large disparity between the enthalpies of duplex disruption and duplex formation as reflecting differences in the single-stranded structures at 25-degrees-C (the initial states in the isothermal mixing experiment) and the single-stranded structures at almost-equal-to 80-degrees-C (the final states in the DSC experiment). In fact, the enthalpy for duplex formation at 25-degrees-C (-56.4 kcal/mol) can be combined with the sum of the integral enthalpies required to melt each single strand from 25 to 80-degrees-C (23.6 kcal/mol for S1 and 27.2 kcal/mol for S2) to calculate a DELTA-H-degrees of -107.2 kcal/mol for the hypothetical process of duplex formation from "random-coil" "unstacked" single strands at 25-degrees-C. The magnitude of this predicted DELTA-H-degrees value for duplex formation is in good agreement with the corresponding parameter we measure directly by DSC for duplex disruption (117.0 kcal/mol), thereby lending credence to our interpretation and analysis of the data. Thus, our results demonstrate that despite being only marginally stable at 25-degrees-C, single strands can exhibit intramolecular interactions that enthalpically poise them for duplex formation.For the duplex studied herein, prior to association at 25-degrees-C, the two complementary single strands already possess > 40% of the total enthalpy (50.8/117) that ultimately stabilizes the final duplex state. This feature of single-stranded structure near room temperature can reduce significantly the enthalpic driving force one might predict for duplex formation from nearest-neighbor data, since such data generally are derived from measurements in which the single strands are in their random-coil states. Consequently, potential contributions from single-stranded structure must be recognized and accounted for when designing hybridization experiments and when using isothermal titration and/or batch mixing techniques to study the formation of duplexes and higher-order DNA structures (e.g., triplexes, tetraplexes, etc.) from their component single strands.