Light-induced formation of G-quadruplex DNA secondary structures
Light-induced formation of G-quadruplex DNA secondary structures
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DOI：
10.1002/cbic.200500198
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发表时间：
2005-11-01
期刊：
影响因子：
3.2
通讯作者：
Heckel, A
中科院分区：
文献类型：
--
作者：
Mayer, G;Kröck, L;Heckel, A
最近，我们开始制备 DNA 和 RNA 衍生物，它们的核碱基上带有笼蔽基团，这可以防止它们形成沃森-克里克碱基对。与选择主要笼罩 DNA [4] 和 RNA 的主链磷酸基团的其他团队相比，[2, 5] 我们希望在特定位点 [6] 引入修饰（理想情况下在任何给定序列中），并生产已确定身份的清洁产品，以获得更好的开/关行为。这是因为事实证明，并非所有笼状修饰都是不活动的，并且并非不同位置的所有修饰都可以同样轻松地删除。为此，我们首先制备了一种含有对光不稳定的 2-(2-硝基苯基)-丙基 (NPP) 的笼状胸苷类似物 (TNPP)，并用它来局部破坏 DNA 双链的稳定性，从而在光触发后可以进行转录。 [7]然后，我们使用相同的残基在空间上阻断蛋白质与适体的相互作用，从而使其功能可光触发。 [8]在这项研究中，我们扩展了笼状脱氧核苷的范围，将笼状鸟苷 (dGNPP) 包括在内，并用它来用光触发高度有序的核酸二级结构的形成。 [9] DNA 或 RNA 分子中的连续 G 核苷酸已知可形成稳定的 G 四链体结构，该结构由 Watson-Crick 和 Hoogsteen 氢键和中心单价阳离子形成（图 1a）。这种核酸排列可以在自然界中发现，并被认为在多种生物过程中发挥重要作用，包括端粒活性的调节、[10] HIV 感染、[11] 以及 HIV-1 整合酶 [12] 和人核拓扑异构酶 1 [13] 的活性。除了染色体端粒区域外，还发现四链体结构位于 c-myc 基因的启动子区域 [14] 和免疫球蛋白开关区域 [15]，它们可能参与基因表达的调节。 [16]人工设计或体外选择的 G-四链体分子已被发现与特定蛋白质紧密相互作用，例如人 α-凝血酶、[17] STAT3 蛋白 [18] 和核仁素。 [19]它们已成功用作同源蛋白功能的拮抗剂。 G-四链体分子也被研究作为小分子药物的潜在靶位点，[20, 21]，例如，抑制端粒酶活性，端粒酶活性在约 85% 的所有癌症中上调。[22]为了用光触发 G-四链体的形成，我们制备了笼状亚磷酰胺 6（方案 1）。从受保护的脱氧鸟苷 (1) 开始，引入异丙基苯氧基乙酰基以保护环外氨基 (→ 2)。该基团可用于“超温和”保护基策略[23]，并且可以例如在室温下用稀氨水去除。我们选择该基团是因为它与之前引入的 TNPP 残基兼容 [7]，并且 NPP 基团也可以在常用的 DNA 固相合成脱保护方案（浓氨水，658C）中裂解。 [24]胺保护后，在 Mitsunobu 条件下引入 NPP 基团 (→ 3)。脱保护 (→ 4) 并在 5’-位 (→ 5) 掺入 DMTr 基团后，可以引入亚磷酰胺 (→ 6)。用于研究端粒的常见模型序列 (7) 是 d (AGGG (TTAGGG) 3)，它源自人类端粒 DNA。 [21]该序列已被证明可折叠成 G-四链体结构，如图 1b 所示。[25]最近在各种缓冲条件（高 K+ 浓度）下观察到不同的 G-四链体结构。 [26]为了
Recently, we have started to prepare DNA and RNA derivatives that bear caging groups on their nucleobases, which prevent them from forming Watson–Crick base pairs. In contrast to other groups that have chosen to mainly cage backbone phosphate groups of DNA [4] and RNA,[2, 5] we want to introduce the modifications at specific sites [6]—ideally in any given sequence—and produce clean products of established identity for a better on/off behaviour. This is because it turns out that not all caged modifications are inactive and not all modifications in different positions can be removed with equal ease. To this end, we first prepared a caged analogue of thymidine (TNPP) that contained a photolabile 2-(2-nitrophenyl)-propyl group (NPP) and used this to locally destabilize a DNA double strand, which could be transcribed after triggering with light.[7] We then used the same residue to sterically block the interaction of a protein with an aptamer and thus made its function light-triggerable.[8] In this study, we have expanded the repertoire of caged deoxynucleosides to include a caged guanosine (dGNPP) and have used it to trigger the formation of highly ordered nucleic-acid secondary structures with light.[9] Consecutive G-nucleotides in DNA or RNA molecules are known to form stable G-quadruplex structures that are created by Watson–Crick and Hoogsteen hydrogen bonding and a central monovalent cation (Figure 1a). Such nucleic acid arrangements can be found in nature and are suggested to play fundamental roles in several biological processes including modulation of telomere activity,[10] HIV infection,[11] and the activity of HIV-1 integrase [12] and human nuclear topoisomerase 1.[13] Besides telomeric regions of chromosomes, quadruplex structures have also been found to be localized in the promoter region of the c-myc gene [14] and in the immunoglobulin switch region [15] where they might be involved in the regulation of gene expression.[16] Artificially designed or in vitro selected G-quadruplex molecules have been found to interact tightly with defined proteins, such as human α-thrombin,[17] STAT3 protein [18] and nucleolin.[19] They have been successfully used as antagonists of the cognate-protein function. G-quadruplex molecules have also been investigated as potential target sites for small-molecule drugs,[20, 21] for example, to inhibit telomerase activity which is up-regulated in about 85% of all cancers.[22]In order to trigger the formation of G-quadruplexes with light we prepared the caged phosphoramidite 6 (Scheme 1). Starting with the protected deoxyguanosine (1) an isopropylphenoxyacetyl group was introduced to protect the exocyclic amino group (→ 2). This group can be used in the “ultramild” protecting-group strategy [23] and can be removed, for example, with dilute ammonia at RT. We chose this group because it is compatible with the previously introduced TNPP residue [7] and the NPP group can also be cleaved in the usual DNA solidphase synthesis deprotection protocol (concentrated ammonia, 658C).[24] After protection of the amine, the NPP group was introduced under Mitsunobu conditions (→ 3). After deprotection (→ 4) and incorporation of the DMTr-group in the 5’-position (→ 5) the phosphoramidite could be introduced (→ 6). A common model sequence (7) for studying telomeres is d (AGGG (TTAGGG) 3), which is derived from human telomeric DNA.[21] This sequence has been shown to fold into the G-quadruplex structure as represented in Figure 1b.[25] Under various buffer conditions (high K+ concentration) different G-quadruplex structures have recently been observed.[26] In order