Photocontrol of single-chain DNA conformation in cell-mimicking microcompartments
Photocontrol of single-chain DNA conformation in cell-mimicking microcompartments
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DOI：
10.1002/cbic.200800072
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发表时间：
2008-05-23
期刊：
影响因子：
3.2
通讯作者：
Baigl, Damien
中科院分区：
文献类型：
--
作者：
Sollogoub, Matthieu;Guieu, Samuel;Baigl, Damien
基因活性的调节强烈依赖于基因组DNA分子的高级结构，这一点已经得到很好的证实。[1]因此，已经开发了几种策略来控制长DNA分子的高阶结构。它们中的大多数是基于使用与DNA结合以中和其电荷的化合物，例如聚胺、多价金属阳离子、阳离子表面活性剂、阳离子聚合物、纳米颗粒或拥挤剂（crowding agent）例如亲水性聚合物。[2]根据这些添加剂的浓度，DNA呈现折叠或未折叠构象。然而，所有这些策略都不可能在恒定的化学组成下以可逆的方式作用于DNA高阶结构。此外，对于转染应用，压实DNA是允许DNA进入细胞的必要步骤。然而，在大多数情况下，DNA在细胞内保持紧凑的构象，这可以显著改变DNA基因表达。因此，使用外部刺激来控制细胞大小的隔间内的DNA高阶结构已成为一个重要的挑战。另一方面，受DNA载体化[3]、人工细胞[4]或生化微反应器[5]制备的影响，许多科学家试图将DNA封装到细胞样微区室中，例如细胞大小的脂质体[6]或磷脂包被的微滴。[7]因此，已经提出了各种成功的策略来制备DNA-脂质体复合物[8]或将DNA包封在脂质体内。[9]在大多数情况下，封装的DNA分子通常小于几千个碱基对。然而，在自然界中，基因组DNA分子可以大得多，高达数百kbp（千碱基对）。据我们所知，还没有提出以受控的方式有效地将大于1kbp的DNA分子包封到细胞大小的脂质体中而不降解的方法。一篇论文报道了T4 DNA分子的包封，但数据不足以得出关于包封DNA链完整性的结论。[10]另一种策略是将DNA封装在紧凑的状态下，但DNA分子一旦被封装就保持其紧凑的状态。[11]最近，Le Ny和Lee提出了一个突破性的系统，在这个系统中，DNA的高级结构可以通过光以可逆的方式控制。[12]这是通过向DNA溶液中加入光敏阳离子表面活性剂偶氮苯三甲基溴化铵表面活性剂（AzoTAB）来实现的。表面活性剂的非极性尾部含有偶氮基团，其在可见条件下主要处于反式（更疏水）构象。在紫外光照射（365 nm）下，偶氮基团光异构化为顺式（更亲水）构象。他们证明了存在AzoTAB浓度范围，在该浓度范围内，DNA在黑暗/可见光条件下处于紧密状态，但在UV照射下处于未折叠状态，即DNA高阶结构可由光控制。在这项研究中，作者主要表征了溶液中多个DNA链的平均性质。在这里，我们的特点是单链构象行为的长基因组DNA作为一个功能的AzoTAB浓度和时间的紫外线照射。我们建立了过渡具有一阶字符在单链水平。我们研究了单链DNA在紫外光照射下的解折叠，证明了单链DNA解折叠的两种机制。然后，我们应用这种策略来展开封装在细胞模拟中的基因组DNA分子。
It has been well established that the regulation of gene activity is strongly dependent on the higher-order structure of genomic DNA molecules.[1] Several strategies have thus been developed to control the higher-order structure of long DNA molecules. Most of them have been based on the use of chemical compounds that bind to DNA to neutralize its charge, such as polyamines, multivalent metal cations, cationic surfactants, cationic polymers, nanoparticles, or crowding agents such as hydrophilic polymers.[2] Depending on the concentration of these additives, DNA exhibits a folded or unfolded conformation. Nevertheless, with all these strategies, it is impossible to act in a reversible way on the DNA higher-order structure under a constant chemical composition. Moreover, for transfection applications, compacting DNA is an essential step to allow the entry of DNA into the cell. In most cases, however, DNA remains in a compact conformation inside the cell, which can significantly alter the DNA gene expression. Using an external stimulus to control DNA higherorder structure within a cell-sized compartment has thus became an important challenge. On the other hand, motivated by the perspective of DNA vectorization,[3] preparation of artificial cells [4] or biochemical microreactors,[5] many scientists have attempted to encapsulate DNA into cell-like microcompartments, for example, cellsized liposomes [6] or phospholipid-coated microdroplets.[7] Consequently, various successful strategies have been proposed to prepare DNA–liposome complexes [8] or encapsulate DNA inside liposomes.[9] In most cases encapsulated DNA molecules were typically smaller than a few thousands base pairs. However, in nature, genomic DNA molecules can be much larger, up to hundreds of kbp (kilo base pairs). To the best of our knowledge, no method has been proposed to encapsulate efficiently, in a controlled way, and without degradation, DNA molecules that are larger than 1 kbp into cell-sized liposomes. One paper reported the encapsulation of T4 DNA molecules, but the data were not sufficient to draw conclusions about the integrity of encapsulated DNA chains.[10] Another strategy was to encapsulate DNA in a compact state, but DNA molecules remained in their compact state once they were encapsulated.[11] Very recently, Le Ny and Lee made a breakthrough by proposing a system where DNA higher-structure can be controlled by light in a reversible manner.[12] This was achieved by adding to a DNA solution a photosensitive cationic surfactant, azobenzene trimethylammonium bromide surfactant (AzoTAB). The apolar tail of the surfactant contains an azo group, which is mainly in the trans (more hydrophobic) conformation under visible conditions. Under UV illumination (365nm), the azo group photoisomerizes into the cis (more hydrophilic) conformation. They demonstrated that there exists an AzoTAB concentration range for which DNA is in the compact state under dark/visible conditions but in the unfolded state under UV illumination, that is, DNA higher-order structure can be controlled by light. In this study, the authors mainly characterized the average property of many DNA chains in solution. Here, we characterized the single-chain conformational behavior of long genomic DNA as a function of AzoTAB concentration and time of UV illumination. We established that the transition has a first-order character at the single-chain level. We studied the single-chain unfolding upon UV illumination and evidenced two mechanisms of single-chain DNA unfolding. Then we applied this strategy to unfold genomic DNA molecules that are encapsulated in cell-mimicking …