Experimental measurement of aromatic stacking affinities in the context of duplex DNA
Experimental measurement of aromatic stacking affinities in the context of duplex DNA
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DOI：
10.1021/ja961733f
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发表时间：
1996-08-28
影响因子：
15
通讯作者：
Kool, ET
中科院分区：
文献类型：
--
作者：
Guckian, KM;Schweitzer, BA;Kool, ET
芳香族分子间的非共价相互作用被广泛认为是生物大分子有序结构稳定的重要因素。1，2最重要的芳香族-芳香族相互作用是在螺旋核酸结构中发现的。由于给定碱基对的最近邻居的身份是DNA双链体中热力学的最佳单一预测因子，因此很明显，芳香族π-π相互作用对这些结构的稳定至关重要。[4]虽然已经有相当多的理论研究旨在模拟DNA中的π-π相互作用[5]，但专门针对DNA本身中堆积（与碱基配对分开）的热力学的实验研究非常少。[6]因此，我们在双链DNA的背景下进行了芳香堆积的研究，我们希望开始阐明稳定这种有序结构的重要力量是什么。我们在这里报告的第一个实验比较自然的DNA碱基和非天然的芳香类似物的堆叠能力的双链DNA。为了将堆叠与双链体DNA中的配对（氢键）相互作用分开，我们将感兴趣的天然或非天然核苷酸放置在碱基配对双链体末端的“悬挂”位置（没有配对伴侣）（图1）。7通过悬挂碱基产生的双链体的稳定性可以通过热变性实验测量，与缺乏添加的核苷酸的双链体进行比较。这种局部电荷所产生的静电效应与芳香堆积的稳定性和几何形状都有关系。为了检验这种效应，我们不仅比较了天然DNA碱基，还比较了具有相似形状和表面积的非极性分子。因此，我们将DNA碱基胸腺嘧啶（1）和腺嘌呤（3）与它们各自的非极性电子等排体二氟甲苯（2）和4-甲基吲哚（4）进行了比较。9我们还比较了芳香烃苯（5）、萘（6）、菲（7）和芘（8）的堆积。已经报道了这些核苷类似物的合成。10-13在pH 7.0和1 M NaCl条件下进行的热力学测量结果见表1。我们测量了作为双链体浓度的函数的解链转变（Tm），并通过绘制1/Tm与ln（[寡核苷酸]）来计算热力学参数。线性拟合相当好（r2 g 0.97），自由能误差约为2%。在这些条件下，未取代的核心双链体的Tm（5 µM）为41.0（（0.5）C），自由能（37 C）为-8.05-（（0.16）kcal/mol。对具有悬挂胸腺嘧啶和腺嘌呤残基的双链体的测量表明，嘌呤在双链体上的堆积比较小的嘧啶碱基更强，这也许并不令人惊讶。两个未配对的脱氧腺苷为自身互补序列增加了2.0 kcal的稳定相互作用，胸腺嘧啶为双链体稳定性增加了1.1 kcal。这种相对堆积能力是从最近邻parameters 3预测的，并且与在RNA中进行的悬挂末端研究一致。7有趣的是，数据显示非极性DNA碱基模拟物的堆叠比它们的天然对应物更强烈。二氟甲苯使双链体的Tm升高了13.4 ℃，大约是胸腺嘧啶的两倍，尽管这两种物质的作用不同。
Noncovalent interactions between aromatic molecules are widely believed to be important contributing factors in the stabilization of organized structure in biological macromolecules. 1, 2 Among the most significant aromatic-aromatic interactions are those found in helical nucleic acid structures. Since the identity of the nearest neighbors to a given base pair is the best single predictor of thermodynamics in DNA duplexes, 3 it is clear that aromatic π-π interactions are crucial to the stabilization of these structures. 4 While there have been a considerable number of theoretical studies aimed at modeling the π-π interaction in DNA, 5 there have been remarkably few experimental studies specifically addressing the thermodynamics of stacking (separate from base pairing) in DNA itself. 6 For that reason we have undertaken a study of aromatic stacking in the context of duplex DNA, and we hope to begin to elucidate what are the important forces which stabilize this organized structure. We report here the first experimental comparison of the stacking abilities of natural DNA bases and of nonnatural aromatic analogs in double-stranded DNA. To separate stacking from pairing (hydrogen-bonding) interactions in duplex DNA we placed the natural or nonnatural nucleotide of interest in a “dangling” position (without a pairing partner) at the end of a base-paired duplex (Figure 1). 7 The resulting stabilization of the duplex by the dangling base can be measured by thermal denaturation experiments, with comparison to the duplex lacking the added nucleotide. Electrostatic effects resulting from such localized charge have been implicated both in the stabilization and in the geometry of aromatic stacking. 5 To examine such effects we compared not only natural DNA bases but also nonpolar molecules with similar shape and surface area. Thus, we compared the DNA base thymine (1) and adenine (3) with their respective nonpolar isosteres difluorotoluene (2) and 4-methylindole (4). 9 We also compared the stacking of the aromatic hydrocarbons benzene (5), naphthalene (6), phenanthrene (7), and pyrene (8). The synthesis of these nucleoside analogs has been reported. 10-13 Results of the thermodynamic measurements made at pH 7.0 and 1 M NaCl are presented in Table 1. We measured melting transitions (Tm) as a function of concentration for the duplexes and calculated thermodynamic parameters by plotting 1/Tm vs ln ([oligonucleotide]). The linear fits were quite good (r2 g 0.97), with error in free energies of approximately (2%. The unsubstituted core duplex under these conditions has a Tm (5 µM) of 41.0 ((0.5) C and a free energy (37 C) of-8.05-((0.16) kcal/mol.Measurement of the duplexes with dangling thymine and adenine residues shows, perhaps not surprisingly, that the purine stacks on the duplex more strongly than the smaller pyrimidine base. The two unpaired deoxyadenosines add 2.0 kcal of stabilizing interaction to the self-complementary sequence, and thymines add 1.1 kcal to the duplex stability. This relative stacking ability is as predicted from nearest-neighbor parameters3 and is consistent with dangling-end studies carried out in RNA. 7 Interestingly, the data show that the nonpolar DNA base mimics stack considerably more strongly than their natural counterparts. Difluorotoluene raises the Tm of the duplex by 13.4 C, about twice the effect of thymine, although the two