Constrained G4 structures unveil topology specificity

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology.

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Fig: (A) Schematic representation of constrained DNA structures used in the pull-down assay. (B) Global strategy to identify constrained G4 interacting proteins from human cells. Biotin-functionalized G4-constrained molecules (1a and 2) and the biotin-functionalized duplex-DNA control 8 were individually mixed with a semi-total human protein extract from HeLa cells, then trapped by streptavidin magnetic beads to isolate interacting proteins. Protein identification was obtained from MS-based quantitative proteomic analysis and further characterized by western-blotting (arrow), or directly by western-blotting (dashed arrow). (C) Diagram showing the differential enrichment of human proteins on constrained G4 structures relative to control duplex DNA. G4 enriched proteins refer to proteins found enriched on 1a and/or 2 G4 constructions relative to the duplex control 8. 214 out of 425 proteins found enriched on constrained G4 have been shown to interact with nucleic acids. Differentially interacting proteins were sorted out using a fold change ≥ 2 and p-value < 0.05, allowing to reach a false discovery rate (FDR) inferior to 5% according to the Benjamini–Hochberg procedure.

Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Pipier, A., Devaux, A., Lavergne, T. et al. Constrained G4 structures unveil topology specificity of known and new G4 binding proteins. Sci Rep 11, 13469 (2021). https://doi.org/10.1038/s41598-021-92806-8

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