Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2′-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate. Quantitative proteomic analysis (LC-MS/MS) of the product(s) obtained by photo-cross-linking NCL to this sequence enabled the identification of one contact site corresponding to a 15-amino acid fragment located in helix α2 of RNA binding domain 2 (RBD2), which sheds light on the role of this structural element in G4-loop recognition. Then, the ability of a panel of benchmark G4 ligands to prevent the NCL–G4 interaction was explored. It was found that only the most potent ligand PhenDC3 can inhibit NCL binding, thereby suggesting that the terminal guanine quartet is also a strong determinant of G4 recognition, putatively through interaction with the RGG domain. This study describes the molecular mechanism by which NCL recognizes G4-containing long loops and leads to the proposal of a model implying a concerted action of RBD2 and RGG domains to achieve specific G4 recognition via a dual loop–quartet interaction.