Development of photo-reactive probes to capture G-quadruplex DNA in complex with endogenous proteins and exogenous synthetic ligands

G-四链体（G4）是可在富含鸟嘌呤的DNA区域中形成的核酸二级结构，并且被认为在DNA加工（复制、转录、重组和端粒维持）中起调节作用。在这一系列中，已经鉴定了少量的G4相互作用蛋白（G4蛋白），其在体外结合、稳定或解析G4结构。许多潜在的G4形成序列存在于基因组中，最近G4结构特异性抗体被用于检测哺乳动物细胞核中的G4结构。尽管如此，我们对它们的生物学功能的理解，特别是它们在细胞环境中与蛋白质的相互作用仍然有限，在这里，我们建议开发替代化学探针，不仅要识别G4-DNA结构，而且要识别它们在细胞环境中相互作用的蛋白质。研究蛋白质-或药物-DNA相互作用的一种特权方法依赖于光交联，其导致相互作用伴侣之间形成共价键。两个系列的三功能捕获化合物（Lig-CC和G4-CC），其含有1）G4特异性小分子配体（G4-配体，Lig）以捕获和稳定G4-结构或已知的G4-DNA作为诱饵以募集G4-蛋白（选择性功能），2）光交联基团（反应功能）以稳定与猎物的相互作用（G4-DNA或G4-蛋白）通过共价键形成，和3）将合成生物素残基（分选功能）以分离具有链霉抗生物素蛋白包被的磁珠的交联复合物。这些分子鱼钩将首先用合成的G4-DNA或纯化的G4-蛋白质进行评估。为此，将使用一组三种结构多样且充分表征的人G4-DNA（C-myc、CEB 1和CEB 25），并且已知其与G4-配体（PhenDC 3）和G4-蛋白（核仁素，Pif 1）相互作用。该步骤将用于在特异性和捕获产率方面选择最佳CC，并建立有效的捕获方案。最有前途的Lig-CC将用于从活细胞中捕获G4-DNA。同时，最好的G4-CC将用于从真核细胞核提取物中钓出未知的G4蛋白。将通过DNA测序鉴定分离的G4-DNA，并通过胰蛋白酶片段化和nanoLC-MS/MS鉴定G4-蛋白。重要的是，将在体内验证候选G4-DNA和G4-蛋白的生物相关性。将构建含有G4-DNA插入或G4-蛋白基因缺失的酵母菌株，并在基因组不稳定性试验中进行分析。我们的协同努力旨在提供有效的化学生物学探针，以确认和定位G4-DNA在真核基因组中的形成，并确定新的蛋白质结合到选定的面板的癌基因和小卫星G4-DNA。我们希望这种策略可以直接应用于后续实验中，使用发现的G4-DNA作为诱饵，在类似的捕获实验，并导致更好地了解G4生物学。

G-quadruplexes (G4) are nucleic acid secondary structures that can form in guanine-rich DNA regions and are believed to play regulatory roles in DNA processing (replication, transcription, recombination and telomere maintenance). In this line, a small number of G4-interacting proteins (G4-proteins) have been identified that bind, stabilize or resolve G4-structures in vitro. Numerous potentially G4-forming sequences are present in genomes and recently G4-structure-specific antibodies were used to detect G4-structures in mammalian cells nuclei. Nonetheless, our understanding concerning their biological functions, in particular on their interactions with proteins in a cellular environment remains limited.Here we propose to develop alternative chemical probes not only to identify G4-DNA-structures but also their interacting proteins in a cellular context. A privileged approach to study protein- or drug-DNA interactions relies on photo-crosslinking which results in the formation of a covalent linkage between the interacting partners. Two series of trifunctional Capture Compounds (Lig-CC and G4-CC) containing 1) a G4-specific small-molecule ligand (G4-ligand, Lig) to trap and stabilize G4-structures OR a known G4-DNA as a bait to recruit G4-proteins (selectivity functions), 2) a photo-crosslinking group (reactivity function) to stabilize the interaction to the prey (G4-DNA or G4-protein) by covalent bond formation, and 3) a biotin residue (sorting function) to isolate crosslinked complexes with streptavidin-coated magnetic beads will be synthesized. These molecular fishing-hooks will be initially evaluated with synthetic G4-DNA or purified G4-proteins. To this end a set of three structurally diverse and well-characterized human G4-DNA (C-myc, CEB1 and CEB25) and known to interact with G4-ligands (PhenDC3) and G4-proteins (nucleolin, Pif 1) will be used. This step will serve to select the best CC in terms of specificity and capture yield and establish efficient capture protocols. The most promising Lig-CC will be used to capture G4-DNA from living cells. In parallel, the best G4-CC will be used to fish-out unknown G4-proteins from eukaryotic nuclear extracts. Isolated G4-DNA will be identified by DNA sequencing and G4-proteins by trypsin fragmentation and nanoLC-MS/MS. Importantly, the biological relevance of candidate G4-DNA and G4-proteins will be validated in vivo. Yeast strains containing insertions of G4-DNA or deletions of genes for G4-proteins will be constructed and analyzed in genome instability assays. Our coordinated efforts aim at providing efficient chemical biology probes to confirm and locate the formation of G4-DNA in eukaryotic genomes and to identify novel proteins binding to the selected panel of oncogene and minisatellite G4-DNA. We expect that this strategy can be directly applied in follow-up experiments using discovered G4-DNA as baits in analogous capture experiments and leads to a better understanding of the G4 biology.