A synthetic block copolymer regulates S1 nuclease fragmentation of supercoiled plasmid DNA.
A synthetic block copolymer regulates S1 nuclease fragmentation of supercoiled plasmid DNA.
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DOI：
10.1002/anie.200500201
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发表时间：
2005-06
影响因子：
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通讯作者：
K. Osada;Y. Yamasaki;S. Katayose;K. Kataoka
中科院分区：
文献类型：
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作者：
K. Osada;Y. Yamasaki;S. Katayose;K. Kataoka
本研究中使用的嵌段共聚物包含阳离子PLL片段（其为质粒结合部分）和非离子PEG片段（其在PLL和质粒DNA之间的离子配对复合物周围形成亲水性和水合栅栏）（参见支持信息）。这允许在没有沉淀的情况下获得水溶性纳米级组装体（100-nm尺寸）。在酸性pH值为4.9的复合物中，特别是在单位摩尔比（氨基/磷酸盐）为1.0时，观察到S1核酸酶对大小特异性质粒DNA的切割。如图1a的泳道2所示，pBR 322 DNA被切割成7个不同的片段（如图1 B中所列）。这种规则的片段化不仅对pBR 322有特异性，而且似乎对其他质粒也很常见。令人惊讶的是，当与PEG-PLL复合时，其他质粒pGL 3-Luc和ColE 1在针对每个原始质粒进行测量时也被切割成相同级分的七个片段（图1a，泳道3和4，以及图1b）。片段化结果的一致性清楚地表明质粒DNA以明确定义的间隔系统裂解。应该注意的是，与PEG-PLL复合的质粒DNA的这种规则切割仅在超螺旋DNA形式中观察到。通过用拓扑异构酶I处理制备的松弛开环DNA也与PEG-PLL形成稳定的复合物，但对S1核酸酶不敏感（数据未显示）。此外，通过用在质粒上具有独特识别位点的限制性酶（EcoRI）处理制备的具有线性质粒DNA的复合物以非特异性方式被顺利降解为寡聚DNA片段（数据未显示）。S1核酸酶对质粒异构体结构的敏感性的差异表明，嵌段共聚物-DNA超分子复合物的拓扑特征影响酶促片段化过程。DNA和阳离子化合物之间形成的聚离子复合物可以诱导螺旋-球状转变，从而导致具有有序形态的凝聚复合物，主要是环形或棒状形式。[14-18]与此一致，电荷中和DNA与PEG-PLL的复合会产生从扩展的DNA超螺旋到紧凑状态的可检测转变，这一点通过静态和动态光散射[13]以及直接观察得到证实。荧光显微镜。[18]虽然在这个复杂的凝聚质粒的结构细节还没有得到澄清，显着的结构特征可能是存在于超螺旋双链DNA后，聚离子诱导的缩合。双链DNA结构中受约束的结构有序（或规则无序）可能在这种聚离子诱导的缩合过程中有规律地重复。这将允许DNA采用特定的结构，以补偿复合过程中伴随构象转变的结构限制。因此，在凝聚的DNA链中的这些特定的无序位点可能优先促进S1核酸酶攻击，从而导致观察到的规则的DNA片段化。
The block copolymer used in this study comprises a cationic PLL segment, which is the plasmid-binding portion, and a nonionic PEG segment, which forms a hydrophilic and hydrated palisade surrounding the ion-paired complex between PLL and plasmid DNA (see the Supporting Information). This allows to obtain a water-soluble nanoscale assembly (100-nm size) without precipitation. The sizespecific plasmid DNA cleavage by S1 nuclease was observed at an acidic pH value of 4.9 for the complex, particularly in a unit molar ratio (amino group/phosphate) of 1.0. As seen in lane 2 of Figure 1a, pBR322 DNA is cut into seven distinct fragments (as listed in Figure 1 b). This regular fragmentation was not specific only for pBR322 but seems common for other plasmids. Surprisingly, the other plasmids, pGL3-Luc and ColE1, when complexed with PEG-PLL, were also cut into seven fragments of the same fractions when measured against each original plasmid (Figure1a, lanes3 and 4, and Figure 1b). The consistency of the fragmentation results clearly indicates the systematic cleavage of plasmid DNA at welldefined intervals. It should be noted that this regular cleavage of plasmid DNA complexed with PEG-PLL is only observed for the supercoiled DNA form. Relaxed open circular DNA, prepared by treatment with topoisomeraseI, also formed stable complexes with PEG-PLL but exhibited no sensitivity to S1 nuclease (data not shown). Additionally, a complex with linear plasmid DNA, prepared by treatment with a restriction enzyme (EcoRI) having a unique recognition site on the plasmid, was smoothly degraded to oligo-DNA pieces in a nonspecific manner (data not shown). The observed differences in S1 nuclease sensitivity to plasmid isomer constructs suggest that topological features of the block copolymer–DNA supramolecular complex influence the enzymatic fragmentation process.Polyion complex formation between DNA and cationic compounds is known to induce a coil–globule transition, thereby resulting in condensed complexes with ordered morphologies, mainly in toroidal or rod-like forms.[14–18] Consistent with this, chargeneutralizing DNA complexation with PEG-PLL produces a detectable transition from an expanded DNA superhelix into a compact state, as confirmed by static and dynamic light scattering [13] as well as by direct observation with fluorescence microscopy.[18] Although the structural details of the condensed plasmid in this complex have not yet been clarified, significant structural features are probably present in the supercoiled double-stranded DNA upon polyion-induced condensation. Constrained structural order (or regular disorder) in the double-stranded DNA structure might be regularly repeated in this polyioninduced condensation process. This would permit the DNA to adopt particular structures that compensate for the structural constraints accompanying conformation transitions during complexation. Consequently, these specific disordered sites in the condensed DNA strands may preferentially promote S1 nuclease attack, thereby resulting in the observed regular DNA fragmentation.