Olsen Chris M, Lee Hui-Ting, Marky Luis A
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.
J Phys Chem B. 2009 Mar 5;113(9):2587-95. doi: 10.1021/jp806853n.
G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T(M)s) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T(M)s in the range from 43.2 to 56.5 degrees C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.
G-四链体是一种经过深入研究的DNA基序，在多种细胞过程中具有潜在作用，最近在衰老和抗癌研究中被视为药物治疗的新靶点。在这项工作中，我们研究了环对G-四链体稳定形成的热力学贡献。具体而言，我们结合使用紫外、圆二色性（CD）和荧光光谱，以及差示扫描量热法（DSC）来确定热力学曲线，包括离子和水的差异结合，用于凝血酶适体：d(GGT2GGTGTGGT2GG)（称为G2）的解折叠。斜体序列TGT和T2已知会形成环。所研究的其他序列在TGT环（TAT、TCT、TTT、TAPT和UUU）、T2环（T4、U2）或两个环（UGU和U2、UUU和U2）中含有碱基替换。所有分子的CD光谱在292 nm处显示一个正峰，这对应于“椅式”构象。每个G-四链体的紫外和DSC熔解曲线显示单相转变，转变温度（Tm）随链浓度增加而保持恒定，证实了它们的分子内形成。这些G-四链体在43.2至56.5摄氏度范围内解折叠，吸热焓为22.9至37.2千卡/摩尔。从每个实验曲线中减去G-四重体堆积的贡献表明，环的存在通过有利的焓贡献、更大的K+离子差异结合（0.1 - 0.6摩尔K+/摩尔）和水分子的可变吸收/释放（-6至8摩尔H2O/摩尔）来稳定每个G-四链体。根据环堆积（环内碱基 - 碱基堆积）及其水合作用讨论了这些特定碱基替换的热力学贡献。
