Department of Chemistry, Kent State University, Kent, Ohio 44242, USA.
J Am Chem Soc. 2010 Jul 7;132(26):8991-7. doi: 10.1021/ja100944j.
Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double-stranded DNA. Whereas single-molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C)-rich region complementary to G quadruplex-forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5'-(TGTCCCCACACCCC)(2), a predominate variant in the insulin-linked polymorphic region (ILPR). We have observed two species with the change in contour length (DeltaL) of 10.4 (+/-0.1) and 5.1 (+/-0.5) nm, respectively. Since DeltaL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br(2) footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23% to 1.3%) with pH 5.5 --> 7.0, which is a well-established behavior for i-motifs. In contrast to that of the i-motif, the formation of the second species with DeltaL of 5.1 nm remains unchanged (6.1 +/- 1.6%) in the same pH range, implying that pH-sensitive C:CH(+) pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the DeltaG(unfold) of an i-motif (16.0 +/- 0.8 kcal/mol), the decreased free energy in the partially folded structure (DeltaG(unfold) 10.4 +/- 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH(+) pairs. Both DeltaL and DeltaG(unfold) argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of an i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.
研究 i 型发夹结构对于全面了解双链 DNA 中 G 四链体的生物学功能非常重要。虽然单分子方法可以从体相技术无法获得的角度描绘 G 四链体，但在富含胞嘧啶 (C) 的与 G 四链体形成序列互补的区域中，关于 i 型发夹结构的类似文献却很少。在这里，我们使用激光镊子研究了 5'-(TGTCCCCACACCCC)(2)（胰岛素连接多态性区域 (ILPR) 中的主要变体）中形成的结构。我们观察到两种具有不同轮廓长度 (DeltaL) 的物种，分别为 10.4(+/-0.1)nm 和 5.1(+/-0.5)nm。由于 10.4nm 的 DeltaL 位于预期的 i 型发夹结构范围内，因此我们将该物种分配给 i 型发夹结构。通过 Br(2)足迹、圆二色性和热变性等体相实验证实了相同序列中 i 型发夹结构的形成。随着 pH 值从 5.5 升高到 7.0，这种结构的形成减少了（从 23%减少到 1.3%），这是 i 型发夹结构的一种公认行为，进一步证实了 i 型发夹结构的形成。与 i 型发夹结构形成的情况相反，在相同的 pH 范围内，具有 5.1nmDeltaL 的第二种物种的形成保持不变（6.1(+/-1.6%），这意味着 pH 敏感的 C:CH(+) 对可能不像 i 型发夹结构那样显著地参与到结构中。与 i 型发夹结构的解折叠自由能 (DeltaG(unfold) 16.0(+/-0.8kcal/mol)相比，部分折叠结构的自由能降低 (DeltaG(unfold) 10.4(+/-0.7kcal/mol)可能反映了一个结构较弱的状态，其中减少了 C:CH(+) 对。DeltaL 和 DeltaG(unfold) 都表明与 i 型发夹结构相比，部分折叠结构具有中间性质。根据这一论点，我们已经通过部分折叠结构直接观察到了 i 型发夹结构的解折叠。i 型发夹结构和部分折叠结构的断裂力相似，均为 22-26pN，高于可阻止 RNA 聚合酶催化的转录的力。这表明，仅从机械角度来看，这些结构中的任何一个都可以阻止 RNA 转录。
