Effective Chiral Discrimination of Tetravalent Polyamines on Single-DNA Compaction
Effective Chiral Discrimination of Tetravalent Polyamines on Single-DNA Compaction
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四价多胺对单 DNA 压缩的有效手性辨别
DOI：
10.1002/anie.201209144
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Tsunehiko Higuchi
中科院分区：
文献类型：
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作者：
Yuko Yoshikawa;Naoki Umezawa;Yuki Imamura;Toshio Kanbe;Nobuki Kato;Kenichi Yoshikawa;Tadayuki Imanaka;Tsunehiko Higuchi
许多体外研究表明，天然和合成多胺与DNA的结合诱导DNA的凝聚/压实。[1-6]这一现象引起了生物学和化学的兴趣，因为基因组DNA通常处于不同程度的缩合状态，并且需要多胺来采用和稳定其紧凑的结构。[1,7]在天然多胺中，精胺（4+）在促进DNA压缩方面比腐胺（2+）和亚精胺（3+）更有效；这一观察结果表明，价态对DNA的压实作用很大几项关于四价多胺的系统研究表明，它们对DNA压实的影响取决于带正电的氨基的几何排列。[6,8,9]除了这些几何效应外，手性效应对DNA压实也有报道。Nayvelt等人通过光散射实验研究了α-甲基化精胺类似物立体异构体的手性对DNA缩聚的影响，发现DNA缩聚所需的阈值浓度有微小但明确的差异；最大差异约为20%在此，我们描述了一种基于四价（4+）多胺立体异构体在单分子DNA中诱导压实的能力，对新合成的立体异构体进行高灵敏度手性鉴别的方法。我们使用的多胺如Scheme1所示。由于许多多胺受体位点，如DNA，是手性的，我们设想在多胺主链中引入立体中心会影响多胺的活性。反式环戊烷环与精胺骨架的简单结合提供了手性和较强的刚性。此外，还引入了对甲氧基苯基作为发色团，用于HPLC纯化时的紫外吸收检测和浓度测定。这些多胺的合成方法类似于先前报道的程序（见支持信息）。[11-13]每种精胺类似物含有两个反式环戊烷单元，共有四个立体中心（方案1）。为了在单分子水平上监测DNA高阶结构的变化，我们使用了一个大的基因组DNA分子，T4 GT7噬菌体DNA （166 kbp, 57 μm）。已经确定，这种大的DNA分子经历了一个离散的构象转变，从膨胀线圈状态到紧致状态在加入各种凝聚剂。[4,14,15]我们通过荧光显微镜观察手性多胺存在和不存在时的大DNA。由于DNA是一个长而薄的分子，并且在体溶液中表现出明显的分子内和平移布朗运动，因此我们采用了倒置宽视场荧光显微镜进行观察
Numerous in vitro studies have shown that the binding of natural and synthetic polyamines to DNA induces the condensation/compaction of DNA.[1–6] This phenomenon is of interest in biology and chemistry, since genomic DNA is often found in different degrees of condensation and requires polyamines for the adoption and stabilization of its compact structures.[1, 7] Among naturally occurring polyamines, spermine (4+) is much more potent at promoting DNA compaction than putrescine (2+) and spermidine (3+); this observation indicates that the valence strongly influences DNA compaction.[4] Several systematic studies on tetravalent polyamines have shown that their influence on DNA compaction is dependent on the geometrical arrangement of positively charged amino groups.[6, 8, 9] As well as such geometric effects, chiral effects on DNA compaction have also been reported. Nayvelt et al. studied the influence of the chirality of stereoisomers of α-methylated spermine analogues on DNA condensation by light-scattering experiments, and showed that there is a small but definite difference in the threshold concentration required for DNA condensation; the maximum difference was approximately 20%.[10] Herein, we describe a method for the highly sensitive chiral discrimination of newly synthesized stereoisomers of tetravalent (4+) polyamines on the basis of their ability to induce compaction in a single molecule of DNA. The polyamines we used are shown in Scheme1. Since many polyamine receptor sites, such as DNA, are chiral, we envisioned that the introduction of stereogenic centers into a polyamine backbone would affect the activity of the polyamine. The simple incorporation of trans-cyclopentane rings into the spermine backbone provided chirality and substantial rigidity. Furthermore, a p-methoxyphenyl group was introduced as a chromophore for the detection of UV absorption during HPLC purification and the convenient determination of concentration. These polyamines were synthesized by a method similar to a previously reported procedure (see the Supporting Information).[11–13] Each spermine analogue contained two trans-cyclopentane units with a total of four stereogenic centers (Scheme 1). To monitor the changes in the higher-order structure of DNA at the single-molecule level, we used a large genomic DNA molecule, T4 GT7 phage DNA (166 kbp, 57 μm). It has been established that such large DNA molecules undergo a discrete conformational transition from an expanded coil state to a compact state upon the addition of various condensing agents.[4, 14, 15] We observed the large DNA in the presence and absence of chiral polyamines by fluorescence microscopy. As DNA is a long, thin molecule and shows significant intramolecular and translational Brownian motions in bulk solution, we adapted an inverted wide-field fluorescence microscope for the observations with a relatively