A caged doxycycline analogue for photoactivated gene expression
A caged doxycycline analogue for photoactivated gene expression
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DOI：
10.1002/anie.200503339
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Cürten, B
中科院分区：
文献类型：
--
作者：
Cambridge, SB;Geissler, D;Cürten, B
条件基因表达范式是研究基因和基因功能的重要工具。然而，目前可用的范例都不允许以高空间和时间分辨率进行转基因表达；相反，它们通常依赖于内源启动子启用的特定表达模式。为了提高转基因表达的分辨率，我们合成了一种光敏（“笼式”）多西环素类似物，用于基于“Tet-on”（Tet=四环素）系统精确光控激活基因。 [1]由于可以轻松且精确地操纵光，这种方法应该能够针对转基因表达的细胞子集，其范围可以从单个细胞和组织斑块到整个器官。 [2]为了实现普遍适用于任何阶段任何生物体的光激活基因表达系统，我们的方法基于条件基因表达范式，该范式使用小的膜渗透分子进行诱导。迄今为止，最著名的诱导系统是 Tet 系统，它通常用于在细胞培养物、组织和整个生物体中诱导转基因。 [1] Tet 系统最有效的类似物是多西环素，它可以与与转录激活结构域 (rtTA) 融合的 Tet 阻遏物的修饰和突变版本结合，并在 Tet 启动子的控制下诱导转基因。 [3]由于多西环素（方案 1）具有多个可衍生化的官能团，因此有必要专门针对转录活性必需的基团，并通过用光敏保护基团“笼罩”来阻断该活性。先前表明，酚类 β-二酮系统（以红色突出显示，方案 1）对于多西环素-镁复合物的形成非常重要，该复合物以高亲和力与 rtTA 蛋白结合。 [4]因此，我们尝试从市售的盐酸多西环素（盐酸半乙醇半水合物）中生成多西环素的 1-（4, 5-二甲氧基-2-硝基苯基）乙基（DMNPE）醚（DMNPE 笼状多西环素），其中 DMNPE 部分连接在酚醛 β-二酮系统上。
Conditional gene-expression paradigms are crucial tools for the study of genes and gene functions. However, none of the currently available paradigms permits transgene expression with high spatial and temporal resolution; rather, they usually rely on specific expression patterns enabled by endogenous promoters. To improve the resolution of transgene expression, we synthesized a photosensitive (“caged”) doxycycline analogue for precise light-controlled activation of genes based on the “Tet-on”(Tet= tetracycline) system.[1] Because of the ease and precision with which light can be manipulated, this approach should make it possible to target subsets of cells for transgene expression which can range from single cells and tissue patches to whole organs.[2] To implement a photoactivated gene-expression system that is generally applicable in any organism at any stage, we based our approach on a conditional gene-expression paradigm that uses a small, membrane-permeant molecule for induction. The most prominent inducible system by far is the Tet system, which is commonly used for the induction of transgenes in cell culture, tissues, and whole organisms.[1] The most potent analogue for the Tet system is doxycycline, which can bind to a modified and mutated version of the Tet repressor fused to a transcriptional activation domain (rtTA) and induce transgenes under the control of the Tet promoter.[3] Because doxycycline (Scheme 1) has several functional groups that may be derivatized, it was necessary to specifically target a group that is essential for transcriptional activity and block this activity by “caging” with a photosensitive protecting group. It was previously shown that the phenolic β-diketone system (highlighted in red, Scheme 1) is important for the formation of the doxycycline–magnesium complex, which binds to the rtTA protein with high affinity.[4] We therefore attempted to generate a 1-(4, 5-dimethoxy-2-nitrophenyl) ethyl (DMNPE) ether of doxycycline (DMNPE-caged doxycycline) from the commercially available doxycycline hyclate (hydrochloride hemiethanolate hemihydrate) with the DMNPE moiety attached at the phenolic β-diketone system.