Targeting nucleolin by RNA G-quadruplex-forming motif

A high level of nucleolin (NCL) expression is often associated with a poor prognosis of patients with lung cancer (LC), suggesting that NCL can be used as a possible biomarker. NCL has been shown to display a marked preference for the binding to G-quadruplexes (G4). Here, we investigate the formation of an RNA quadruplex structure in a sequence found in the human precursor pre-MIR150 with the potential to recognize NCL. Circular dichroism (CD) spectra of pre-MIR150 G4-forming sequence (designated by rG4) indicate the formation of a parallel quadruplex structure in KCl or when complexed with the well-known G4 ligand PhenDC3. The thermal stability of rG4 is very high, and further increases in the presence of PhenDC3. The binding affinities of rG4 to PhenDC3 and NCL RBD1,2 are similar with K_D values in the nanomolar range. PAGE results suggest the formation of a ternary quadruplex-ligand-protein complex (rG4-PhenDC3-NCL RBD1,2), indicative that PhenDC3 does not prevent the binding of rG4 to NCL RBD1,2. Finally, rG4 can recognize NCL-positive cells and, when fluorescently labeled, can be used as a probe for this protein. ELISA experiments indicate altered NCL expression patterns in liquid biopsies of LC patients in a non-invasive manner, potentially helping the diagnosis, prognosis, and patient response to treatment. Hence, labeled rG4 could be used as a detection probe of LC in liquid biopsies.