Santos, TiagoMiranda, AndréImbert, LionelJardim, AndreiaCaneira, Catarina R. F.Chu, VirgíniaConde, João PedroCampello, Maria Paula CabralPaulo, AntónioSalgado, GilmarCabrita, Eurico J.Cruz, Carla2022-03-112022-03-112022-02-010928-0987PURE: 36467709PURE UUID: ad19b4dc-2f05-4995-a7ec-cdc3fc9664e9Scopus: 85121779205PubMed: 34922315WOS: 000804547300006http://hdl.handle.net/10362/134341PD/BD/142851/2018 PD/00065/2013 MIT-EXPL/BIO/0008/2017 IF/00959/2015One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K+ ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10−5–10−6 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10−10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10−10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5′-GGGAGGGAGGGACGGG- 3′) and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples.7075072engG-quadruplex ligandsMolecular receptorsNucleolinPre-miRNA-149RNA G-quadruplexPharmaceutical Sciencejournal article10.1016/j.ejps.2021.106093https://www.scopus.com/pages/publications/85121779205