Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies.
Sequence specific and high affinity recognition of 5'-ACGCGT-3' by rationally designed pyrrole-imidazole H-pin polyamides: thermodynamic and structural studies.
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DOI：
10.1016/j.bmc.2008.09.034
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发表时间：
2008-10-15
影响因子：
3.5
通讯作者：
Lee, Moses
中科院分区：
文献类型：
--
作者：
Mackay, Hilary;Brown, Toni;Uthe, Peter B.;Westrate, Laura;Sielaff, Alan;Jones, Justin;Lajiness, James P.;Kluza, Jerome;O'Hare, Caroline;Nguyen, Binh;Davis, Zach;Bruce, Chrystal;Wilson, W. David;Hartley, John A.;Lee, Moses
含有咪唑 (Im) 和吡咯 (Py) 的聚酰胺可形成堆叠二聚体，可通过编程来靶向 DNA 小沟中的特定序列并控制基因表达。尽管已经针对 DNA 序列识别对聚酰胺的各种设计进行了彻底研究，但 H-pin 聚酰胺（共价交联聚酰胺）的使用尚未受到足够的关注。因此，我们设计了实验来系统地研究由 PyImPyIm (5) 或 f-ImPyIm (3e, f = 甲酰胺基) 与乙二醇接头连接组成的两种对称 H-pin 聚酰胺的 DNA 识别特性。这些化合物的创建是为了分别通过重叠和交错的结合基序识别同源 5'-ACGCGT-3'。 DNaseI 足迹、热变性、圆二色性、表面等离子共振和等温滴定微量热法研究的结果表明，两种 H-pin 聚酰胺的结合亲和力比各自的单体更高。含有甲酰胺基的H-pin 3e的结合亲和力比四酰胺H-pin 5的结合亲和力高出一百多倍，这证明了具有甲酰胺基团和交错基序在增强亲和力方面的重要性。然而，与 H-pin 3e 相比，四酰胺 H-pin 5 表现出对同源序列优于其非同源序列 ACCGGT 和 AAATTT 的结合偏好。 SPR 实验数据得出 PyImPyIm H-pin 5 和 f-ImPyIm H-pin 3e 的结合常数分别为 1.6 × 108 M−1 和 2.0 × 1010 M−1。与相应的未连接的 PyImPyIm 4 和 f-ImPyIm 2 对应物相比，两个 H-pin 的结合亲和力显着更高（约 100 倍）。 ITC 分析揭示了 298 K 时的适度反应焓（5 和 3e 的 ΔH 分别为 -3.3 和 -1.0 kcal mol−1），表明这些是熵驱动的相互作用。热容 (ΔCp) 分别确定为 -116 和 -499 cal mol−1 K−1。这些结果与使用与 (5'-CCACGCGTGG)2 结合的 H-pin 复合物根据溶剂可及表面积的变化确定的 ΔCp 值基本一致。根据模型，H 形销钉紧贴在小凹槽中，连接器将两个聚酰胺部分舒适地固定到位，氧原子指向溶剂。总之，H-pin 聚酰胺为发现未来几代能够以高亲和力和选择性结合靶 DNA 序列的可编程小分子提供了重要的分子设计基序。
Imidazole (Im) and Pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific sequences in the minor groove of DNA and control gene expression. Even though various designs of polyamides have been thoroughly investigated for DNA sequence recognition, the use of H-pin polyamides (covalently cross-linked polyamides) has not received as much attention. Therefore, experiments were designed to systematically investigate the DNA recognition properties of two symmetrical H-pin polyamides composed of PyImPyIm (5) or f-ImPyIm (3e, f = formamido) tethered with an ethylene glycol linker. These compounds were created to recognize the cognate 5′-ACGCGT-3′ through an overlapped and staggered binding motif, respectively. Results from DNaseI footprinting, thermal denaturation, circular dichroism, surface plasmon resonance and isothermal titration microcalorimetry studies demonstrated that both H-pin polyamides bound with higher affinity than their respective monomers. The binding affinity of formamido-containing H-pin 3e was more than a hundred times greater than that for the tetraamide H-pin 5, demonstrating the importance of having a formamido group and the staggered motif in enhancing affinity. However, compared to H-pin 3e, tetraamide H-pin 5 demonstrated superior binding preference for the cognate sequence over its non-cognates, ACCGGT and AAATTT. Data from SPR experiments yielded binding constants of 1.6 × 108 M−1 and 2.0 × 1010 M−1 for PyImPyIm H-pin 5 and f-ImPyIm H-pin 3e, respectively. Both H-pins bound with significantly higher affinity (ca. 100-fold) than their corresponding unlinked PyImPyIm 4 and f-ImPyIm 2 counterparts. ITC analyses revealed modest enthalpies of reactions at 298 K (ΔH of −3.3 and −1.0 kcal mol−1 for 5 and 3e, respectively), indicating these were entropic-driven interactions. The heat capacities (ΔCp) were determined to be −116 and −499 cal mol−1 K−1, respectively. These results are in general agreement with ΔCp values determined from changes in the solvent accessible surface areas using complexes of the H-pins bound to (5′-CCACGCGTGG)2. According to the models, the H-pins fit snugly in the minor groove and the linker comfortably holds both polyamide portions in place, with the oxygen atoms pointing into the solvent. In summary, the H-pin polyamide provides an important molecular design motif for the discovery of future generations of programmable small molecules capable of binding to target DNA sequences with high affinity and selectivity.
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DOI：
10.1073/pnas.2035074100
发表时间：
2003-10-14
影响因子：
11.1
作者：
Best, TP;Edelson, BS;Dervan, PB
通讯作者：
Dervan, PB
影响因子：
3.5
作者：
Belitsky, JM;Leslie, SJ;Dervan, PB
通讯作者：
Dervan, PB
影响因子：
3.5
作者：
Brown, Toni;Mackay, Hilary;Lee, Moses
通讯作者：
Lee, Moses
影响因子：
3.4
作者：
Nguyen, B;Stanek, J;Wilson, WD
通讯作者：
Wilson, WD
影响因子：
2.9
作者：
Buchmueller, Karen L.;Bailey, Suzanna L.;Lee, Moses
通讯作者：
Lee, Moses