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

• Applicant: Professor Dr. Elmar Weinhold
• Subject Area: Biological and Biomimetic Chemistry
• Term: from 2014 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 258773983

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.

• DFG Programme: Research Grants
• International Connection: France
• Participating Persons: Dr. Alain Nicolas; Dr. Marie-Paule Teulade-Fichou