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

G-四联体(G4)是一种核酸二级结构，可以在富含鸟嘌呤的DNA区域形成，并被认为在DNA加工(复制、转录、重组和端粒维持)中发挥调节作用。在这个品系中，已经鉴定了少量的G4相互作用蛋白(G4蛋白)，它们在体外结合、稳定或分解G4结构。基因组中存在大量潜在的G4形成序列，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，特别是它们与细胞环境中蛋白质的相互作用的了解仍然有限。在这里，我们建议开发替代的化学探针，不仅可以识别G4-DNA结构，还可以识别它们在细胞环境中的相互作用蛋白。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，这导致相互作用的伙伴之间形成共价键。将合成两个系列的三功能捕获化合物(Lig-CC和G4-CC)，其中包括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作为诱饵以招募G4-蛋白质(选择性功能)，2)光交联基(反应功能)以形成共价键稳定与猎物(G4-DNA或G4-蛋白质)的相互作用，以及3)生物素残基(分类功能)以分离与链霉亲和素包裹的磁珠的交联络合物。这些分子钓鱼钩将首先用合成的G4-DNA或纯化的G4-蛋白进行评估。为此，将使用一套结构多样且特征良好的三种人类G4-DNA(C-myc、CEB1和CEB25)，并已知与G4配体(PhenDC3)和G4蛋白质(核仁蛋白，Pif1)相互作用。这一步骤将有助于从特异性和捕获率方面选择最好的CC，并建立有效的捕获方案。最有希望的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液中筛选未知的G4-蛋白。分离的G4-DNA将通过DNA测序和G4-蛋白的胰酶裂解和纳米LC-MS/MS鉴定，重要的是，候选的G4-DNA和G4-蛋白的生物学相关性将在体内得到验证。含有G4-DNA插入或G4-蛋白基因缺失的酵母菌株将被构建并在基因组不稳定性分析中进行分析。我们的协调工作旨在提供有效的化学生物学探针，以确认和定位真核基因组中G4-DNA的形成，并鉴定与选定的癌基因和小卫星G4-DNA结合的新蛋白。我们期待这一策略可以直接应用于后续实验中，以发现的G4-DNA为诱饵，在类似的捕获实验中，并导致对G4生物学的更好理解。

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.